阅读说明：本技术 pSFV-p32病毒样颗粒及其制备方法和应用 (pSFV-p32 virus-like particle and preparation method and application thereof ) 是由 方倪冉 黄梅 郑航辉 徐婷 杨小云 董楠 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种pSFV-p32病毒样颗粒的制备方法,包括：(1)构建得到pSFV-sp6-p32质粒；(2)将pSFV-sp6-p32、SFV-helper质粒体外转录成mRNA,然后将pSFV-sp6-p32、SFV-helper质粒体外转录的mRNA共电转入BHK细胞中,得到pSFV-p32病毒样颗粒。相应的,本发明还提供上述制得的pSFV-p32病毒样颗粒及其应用。实施本发明,通过插入非洲猪瘟p32基因,制得的pSFV-p32病毒样颗粒能够高效表达外源蛋白p32,有利于非洲猪瘟蛋白的表达筛选和RNA疫苗研制。(The invention discloses a preparation method of pSFV-p32 virus-like particles, which comprises the following steps: (1) constructing and obtaining pSFV-sp6-p32 plasmid; (2) the pSFV-sp6-p32 and SFV-helper plasmids are transcribed into mRNA in vitro, and then the mRNA transcribed in vitro by the pSFV-sp6-p32 and the SFV-helper plasmids are co-electroporated into BHK cells to obtain the pSFV-p32 virus-like particles. Correspondingly, the invention also provides the pSFV-p32 virus-like particle prepared by the method and application thereof. By implementing the invention, the pSFV-p32 virus-like particle prepared by inserting the African swine fever p32 gene can efficiently express the foreign protein p32, and is beneficial to expression screening of the African swine fever protein and development of an RNA vaccine.)

1. A preparation method of pSFV-p32 virus-like particles is characterized by comprising the following steps:

(1) constructing and obtaining pSFV-sp6-p32 plasmid;

(2) transcribing pSFV-sp6-p32 and SFV-helper plasmids into mRNA in vitro, and then co-transfecting the mRNA transcribed in vitro by the pSFV-sp6-p32 and the SFV-helper plasmids into BHK cells to obtain pSFV-p32 virus-like particles;

the pSFV-sp6-p32 plasmid is obtained by taking Semliki forest virus as a vector, synthesizing an ASFV p32 gene shown as SEQ ID NO.1 and inserting the ASFV p32 gene into the vector.

2. The method of preparing pSFV-p32 virus-like particles according to claim 1, wherein the step (1) comprises:

(1.1) synthesizing an ASFV p32 gene, wherein the sequence of the ASFV p32 gene is SEQ ID NO. 1;

(1.2) designing and synthesizing primers tongyong-F and SP6-p32-R, wherein the sequence of the primer tongyong-F is SEQ ID NO.2, and the sequence of the primer SP6-p32-R is SEQ ID NO. 3;

(1.3) carrying out enzyme digestion on the double-enzyme digestion plasmid pSFV-sp6-EGFP by using BamHI/SmaI;

(1.4) carrying out homologous recombination on the ASFV p32 gene fragment synthesized by the gene and the enzyme-cut vector fragment obtained in the step (1.3) to obtain a recombinant product;

(1.5) adding the recombinant product to a competent cell DH5 alpha cell to perform recombinant product transformation;

(1.6) carrying out recombinant product identification on the transformed recombinant product;

(1.7) extracting plasmids from the identified recombinant products;

(1.8) carrying out enzyme digestion identification and sequencing on the plasmid.

3. The method for preparing pSFV-p32 virus-like particles according to claim 2, wherein in step (1.3), the pSFV-sp6-EGFP is prepared by:

designing and synthesizing primers, wherein the primers comprise design primers Cs-EGFP-F and Cs-EGFP-R, and bacterial liquid identification primers EGFP-identification-F and EGFP-identification-R:

amplifying a Cs-EGFP fragment by taking EGFP-C1 as a template and Cs-EGFP-F and Cs-EGFP-R as primers, and then constructing a replicon plasmid pSFVCs-sp6-EGFP through enzyme digestion, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing;

pSFVCs-sp6-EGFP is used as a template, EGFP-F and EGFP-R are used as primers to amplify fragments, and then the replicon plasmid pSFV-sp6-EGFP is constructed by enzyme digestion, homologous recombination, transformation and identification of recombinant products, plasmid extraction, enzyme digestion identification and sequencing.

4. The method of claim 2, wherein the step (1.6) of identifying the recombinant product obtained by transforming the pSFV-p32 virus-like particle comprises:

culturing the transformed recombinant product overnight and selecting bacterial colonies for bacterial liquid identification;

the PCR system identified by the bacterial liquid comprises the bacterial liquid, a designed primer tongyong-F and a designed primer SP6-p 32-R.

5. The method of preparing pSFV-p32 virus-like particles according to claim 1, wherein the step (2) comprises:

(2.1) linearizing pSFV-sp6-p32 and SFV-helper;

(2.2) in vitro transcription of the linearized pSFV-sp6-p32 and SFV-helper;

(2.3) co-transfecting the pSFV-sp6-p32 and the mRNA transcribed in vitro by the SFV-helper into BHK cells to obtain pSFV-p32 virus-like particles.

6. The method of preparing pSFV-p32 virus-like particle according to claim 1 or 5, wherein the SFV-helper plasmid is obtained by constructing a recombinant vector represented by SEQ ID NO.4, synthesizing an insert gene represented by SEQ ID NO.5, and cloning the insert gene into the recombinant vector.

7. The method of claim 6, wherein the SFV-helper plasmid is prepared by the method comprising:

constructing a recombinant vector, wherein the sequence of the constructed recombinant vector is SEQ ID NO. 4;

synthesizing an insert gene, wherein the sequence of the insert is SEQ ID NO. 5;

carrying out homologous recombination on the recombinant vector and the insert fragment, transforming a recombinant product into DH5 alpha competent bacteria, coating a flat plate, carrying out inverted culture, and extracting a plasmid to obtain an SFV-helper plasmid, wherein the sequence of the SFV-helper plasmid is SEQ ID NO. 6.

8. A virus-like particle of pSFV-p32, produced by the method of any one of claims 1 to 7.

9. The preparation method of the pSFV-p32 virus-like particle as claimed in any one of claims 1 to 7 and the use of the pSFV-p32 virus-like particle as claimed in claim 8 in RNA vaccines, therapeutics, animal models.

10. The use according to claim 9, wherein the RNA vaccine is a vaccine for controlling epidemic african swine fever.

Technical Field

The invention relates to the field of bioengineering, in particular to pSFV-p32 virus-like particles and a preparation method and application thereof.

Background

ASFV is a large-scale intracellular replication virus, the susceptible cell is a pig mononuclear-macrophage line, and the destruction of the immune system causes an acute, hot and high-contact infectious disease of pigs, namely African swine fever. African Swine Fever Virus (ASFV) belongs to the order of DNA viruses (dsDNA), the family of African swine fever viruses, the genus African swine fever virus (Asfivirus) in the viral taxonomy, is the only member of the family of African swine fever viruses, with virions in icosahedral symmetry and an average diameter of about 200 nm. The clinical symptoms of African swine fever are mainly characterized by high fever, inappetence, bleeding of skin and internal organs and high mortality, no effective vaccine is available for prevention at present, and once the clinical symptoms are discovered, the vaccine can only be killed on a large scale, so that great loss is caused to the pig industry. The disease belongs to animal epidemic disease which is legally reported by the requirements of the world animal health Organization (OIE), and is classified as animal epidemic disease in the national animal pathogenic microorganism catalogue. The structure of the virus particle is nucleosome, nucleocapsid and envelope (divided into outer layer and inner layer) from inside to outside: the nucleoplasm mainly comprises a viral genome, DNA binding proteins (p10, p14, p37, p34 and the like) and enzymes required for early transcription of genes; the nucleocapsid mainly contains the structural proteins p72 and p17, and consists of approximately 2000 capsomeres; the inner capsule membrane is derived from the endoplasmic reticulum of a host, is combined with p12, p22, p32, p54 and CD2v protein, and the outer capsule membrane is a virus budding subsidiary product and is loosely surrounded. The ASFV genome is a linear double-stranded DNA molecule, the full length of different strains has different sizes, the approximate range is 170-193 kb, and 150-175 proteins are coded. The genome consists of a central conserved region (about 125kb), variable regions on both sides (38-48 kb, 13-22 kb, respectively), and inverted repeats (about 2.1-2.5 kb) at the end of the genome. The molecular weight of the p32 protein studied herein is about 30 kD. The protein is expressed in the early stage of virus infection and can cause the body to generate immune response, so the protein has better antigenicity. The study showed that antibodies against p32 protein could inhibit virus internalization, indicating that p32 protein is also involved in the virus entry process into cells. No report of the onset of African swine fever is found in China before 8 months in 2018. Governments and researchers have never relaxed monitoring of this disease. China joins the global African swine fever union in 2013, and the African swine fever union and members of various countries in the world jointly strengthen the prevention and control work of African swine fever, and develop basic research on ASFV. The deep understanding of the immune response of the body after being infected with the virus has very important significance for the prevention and control of ASF and the research and development of vaccines. In recent 20 years, ASF subunit vaccines and recombinant vaccines are widely researched, and various viral proteins involved in ASFV infection of organisms, such as p72, p32, p54 and the like, become target proteins for research, but currently developed subunit vaccines and recombinant vaccines still cannot generate enough protection force on the organisms, can only reduce the viremia level to a certain extent and delay the occurrence time of clinical symptoms.

The RNA replicon vaccine is a novel vaccine which is based on RNA replicon and can perform autonomous replication. The vaccine is expressed in cytoplasm, cannot integrate with genome, and has good safety. The RNA replicon vaccine can autonomously replicate and induce whole immune cell immunity and mucosal immunity, and has the advantages of high efficiency, wide application range and the like, thereby being attracted by attention. An RNA replicon is an RNA that is derived from the genome of an RNA virus and is capable of autonomous replication. The viruses most commonly used for replicon development are alphaviruses (alphaviruses) in the togaviridae family, such as Sindbis virus (SIN), Semliki Forest Virus (SFV), and Venezuelan equine encephalitis Virus (VEE); in addition to alphaviruses, there are flaviviruses, picornaviruses, paramyxoviruses, caliciviruses, and the like. An RNA replicon expressing an antigen can be delivered by 3 means, (1) naked RNA transcribed in vitro; (2) constructing plasmid DNA which is transcribed into replicon RNA (based on DNA) by cell RNA polymerase II in vivo; (3) the replicon RNA is packaged into a virus-like particle.

Naked RNA has the defects of instability, quick degradation, difficult storage and the like, virus-like particles have no infectivity, and as immunogen, the virus-like particles can be presented to immune cells through the same way as virus infection, effectively induce the immune system of an organism to generate immune protection reaction, and become good vaccine; as a gene therapy vector, the epitope of foreign protein can be inserted, and the purpose of specific therapy can be achieved by releasing the foreign protein; the presence of the corresponding receptor on the cell surface has attracted considerable attention both for its stability and for its efficiency.

Disclosure of Invention

The invention aims to solve the technical problem of providing pSFV-p32 virus-like particles and a preparation method thereof, wherein the pSFV-p32 virus-like particles prepared by inserting African swine fever p32 gene can efficiently express foreign protein p32, and are beneficial to expression screening of African swine fever protein and RNA vaccine development.

The technical problem to be solved by the invention is also to provide application of pSFV-p32 virus-like particles.

In order to solve the technical problems, the invention provides a preparation method of pSFV-p32 virus-like particles, which comprises the following steps:

(1) constructing and obtaining pSFV-sp6-p32 plasmid;

(2) transcribing pSFV-sp6-p32 and SFV-helper plasmids into mRNA in vitro, and then co-transfecting the mRNA transcribed in vitro by the pSFV-sp6-p32 and the SFV-helper plasmids into BHK cells to obtain pSFV-p32 virus-like particles;

the pSFV-sp6-p32 plasmid is obtained by taking Semliki forest virus as a vector, synthesizing an ASFV p32 gene shown as SEQ ID NO.1 and inserting the ASFV p32 gene into the vector.

As an improvement of the technical scheme, the step (1) comprises the following steps:

(1.1) synthesizing an ASFV p32 gene, wherein the sequence of the ASFV p32 gene is SEQ ID NO. 1;

(1.2) designing and synthesizing primers tongyong-F and SP6-p32-R, wherein the sequence of the primer tongyong-F is SEQ ID NO.2, and the sequence of the primer SP6-p32-R is SEQ ID NO. 3;

(1.3) carrying out enzyme digestion on the double-enzyme digestion plasmid pSFV-sp6-EGFP by using BamHI/SmaI;

(1.4) carrying out homologous recombination on the ASFV p32 gene fragment synthesized by the gene and the enzyme-cut vector fragment obtained in the step (1.3) to obtain a recombinant product;

(1.5) adding the recombinant product to a competent cell DH5 alpha cell to perform recombinant product transformation;

(1.6) carrying out recombinant product identification on the transformed recombinant product;

(1.7) extracting plasmids from the identified recombinant products;

(1.8) carrying out enzyme digestion identification and sequencing on the plasmid.

As an improvement of the technical scheme, in the step (1.3), the pSFV-sp6-EGFP is prepared by the following method:

designing and synthesizing primers, wherein the primers comprise design primers Cs-EGFP-F and Cs-EGFP-R, and bacterial liquid identification primers EGFP-identification-F and EGFP-identification-R:

amplifying a Cs-EGFP fragment by taking EGFP-C1 as a template and Cs-EGFP-F and Cs-EGFP-R as primers, and then constructing a replicon plasmid pSFVCs-sp6-EGFP through enzyme digestion, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing;

pSFVCs-sp6-EGFP is used as a template, EGFP-F and EGFP-R are used as primers to amplify fragments, and then the replicon plasmid pSFV-sp6-EGFP is constructed by enzyme digestion, homologous recombination, transformation and identification of recombinant products, plasmid extraction, enzyme digestion identification and sequencing.

As an improvement of the above technical scheme, in the step (1.6), the identification of the recombinant product of the transformed recombinant product comprises the following steps:

culturing the transformed recombinant product overnight and selecting bacterial colonies for bacterial liquid identification;

the PCR system identified by the bacterial liquid comprises the bacterial liquid, a designed primer tongyong-F and a designed primer SP6-p 32-R.

As an improvement of the technical scheme, the step (2) comprises the following steps:

(2.1) linearizing pSFV-sp6-p32 and SFV-helper;

(2.2) in vitro transcription of the linearized pSFV-sp6-p32 and SFV-helper;

(2.3) co-transfecting the pSFV-sp6-p32 and the mRNA transcribed in vitro by the SFV-helper into BHK cells to obtain pSFV-p32 virus-like particles.

As an improvement of the technical scheme, the SFV-helper plasmid is obtained by constructing a recombinant vector shown as SEQ ID NO.4, synthesizing an insert gene shown as SEQ ID NO.5 and cloning the insert gene into the recombinant vector.

As an improvement of the technical scheme, the SFV-helper plasmid is prepared by the following method:

constructing a recombinant vector, wherein the sequence of the constructed recombinant vector is SEQ ID NO. 4;

synthesizing an insert gene, wherein the sequence of the insert is SEQ ID NO. 5;

carrying out homologous recombination on the recombinant vector and the insert fragment, transforming a recombinant product into DH5 alpha competent bacteria, coating a flat plate, carrying out inverted culture, and extracting a plasmid to obtain an SFV-helper plasmid, wherein the sequence of the SFV-helper plasmid is SEQ ID NO. 6.

Correspondingly, the invention also provides pSFV-p32 virus-like particles, which are prepared by the preparation method.

Correspondingly, the invention also discloses application of the pSFV-p32 virus-like particle and a preparation method of the pSFV-p32 virus-like particle in RNA vaccines, therapeutic drugs and animal models.

As an improvement of the technical scheme, the RNA vaccine is a vaccine for preventing and controlling African swine fever epidemic disease.

The implementation of the invention has the following beneficial effects:

the invention adopts Semliki Forest Virus (SFV) as a vector to construct a packaging system, wherein one RNA molecule is an RNA replicon vector containing an inserted ASFV p32 protein. The other RNA molecule is auxiliary RNA, and after the auxiliary RNA is co-electroporated into BHK cells, the supernatant is collected to successfully prepare virus-like particles pSFV-p 32; the protein expression of p32 is successfully identified through IFA experiments, thereby providing reliable experimental basis and scientific basis for the expression screening of other important African foreign proteins and the development of mRNA vaccines, and being beneficial to the development of vaccines for preventing and controlling African swine fever epidemic diseases.

Therefore, the pSFV-p32 virus-like particle can efficiently express foreign protein p32, inhibit the expression of virus structural protein and has higher biological safety.

Drawings

FIG. 1 is a map of a recombinant plasmid pSFV-sp6-p 32;

FIG. 2 is a diagram showing the cleavage result of pSFV-sp 6-EGFP;

FIG. 3 is a schematic diagram showing the identification result of recombinant plasmid bacterial liquid;

FIG. 4 is a schematic diagram showing the restriction and identification results of recombinant plasmid

FIG. 5 is a schematic diagram showing the in vitro transcription result of pSFV-sp6-p 32;

FIG. 6 is a schematic diagram showing the in vitro transcription result of pSFV-helper;

FIG. 7 is a schematic diagram showing the result of IFA detection of ASFV p32 protein expression;

FIG. 8 is a map of the template plasmid pEGFP-C1 for amplification of Cs-EGFP;

FIG. 9 is a map of the pSFVCs-LacZ plasmid;

FIG. 10 is a map of the pSFVCs-sp6-EGFP plasmid.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

The invention provides a preparation method of pSFV-p32 virus-like particles, which comprises the following steps:

(1) constructing and obtaining pSFV-sp6-p32 plasmid;

the pSFV-sp6-p32 plasmid is obtained by taking Semliki forest virus as a vector, synthesizing an ASFV p32 gene shown as SEQ ID NO.1 and inserting the ASFV p32 gene into the vector.

Specifically, the step (1) comprises the following steps:

(1.1) synthesizing an ASFV p32 gene, wherein the sequence of the ASFV p32 gene is SEQ ID NO. 1;

(1.2) designing and synthesizing primers tongyong-F and SP6-p32-R, wherein the sequence of the primer tongyong-F is SEQ ID NO.2, and the sequence of the primer SP6-p32-R is SEQ ID NO. 3;

(1.3) carrying out enzyme digestion on the double-enzyme digestion plasmid pSFV-sp6-EGFP by using BamHI/SmaI;

(1.4) carrying out homologous recombination on the ASFV p32 gene fragment synthesized by the gene and the enzyme-cut vector fragment obtained in the step (1.3) to obtain a recombinant product;

(1.5) adding the recombinant product to a competent cell DH5 alpha cell to perform recombinant product transformation;

(1.6) carrying out recombinant product identification on the transformed recombinant product;

(1.7) extracting plasmids from the identified recombinant products;

(1.8) carrying out enzyme digestion identification and sequencing on the plasmid.

Preferably, the pSFV-sp6-EGFP in the step (1.3) is prepared by the following method:

designing and synthesizing primers, wherein the primers comprise design primers Cs-EGFP-F and Cs-EGFP-R, and bacterial liquid identification primers EGFP-identification-F and EGFP-identification-R:

and amplifying the Cs-EGFP fragment by taking the EGFP-C1 as a template and the Cs-EGFP-F and Cs-EGFP-R as primers, and then constructing and obtaining the replicon plasmid pSFVCs-sp6-EGFP through enzyme digestion, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing.

And thenpSFVCs-sp6-EGFP is used as a template, designed and synthesized primers EGFP-F and EGFP-R are used for amplifying an EGFP fragment, and then the replicon plasmid pSFV-sp6-EGFP is finally constructed through enzyme digestion pSFVCs-sp6-EGFP, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing. Preferably, step (1.4) comprises:

and (3) preparing the following reaction system on ice according to a proper volume by using the ASFV p32 gene fragment obtained by gene synthesis and the vector fragment obtained by enzyme digestion in the step (1.3). The volume ratio of the ASFV p32 gene fragment to the enzyme digestion vector large fragment is 1: 2.

Preferably, in step (1.6), the identification of the recombinant product after transformation comprises:

culturing the transformed recombinant product overnight and selecting bacterial colonies for bacterial liquid identification;

the PCR system identified by the bacterial liquid comprises the bacterial liquid, a designed primer tongyong-F and a designed primer SP6-p 32-R. According to the concentration, the added bacteria liquid, the design primer tongyong-F and the design primer SP6-p32-R have the volume ratio of (1-2) to (1-2).

(2) Transcribing pSFV-sp6-p32 and SFV-helper plasmids into mRNA in vitro, and then co-transfecting the mRNA transcribed in vitro by the pSFV-sp6-p32 and the SFV-helper plasmids into BHK cells to obtain pSFV-p32 virus-like particles;

specifically, the step (2) comprises the following steps:

(2.1) linearizing pSFV-sp6-p32 and SFV-helper;

(2.2) in vitro transcription of the linearized pSFV-sp6-p32 and SFV-helper;

(2.3) co-transfecting the pSFV-sp6-p32 and the mRNA transcribed in vitro by the SFV-helper into BHK cells to obtain pSFV-p32 virus-like particles.

Preferably, in step (2.1), the SFV-helper plasmid is obtained by constructing a recombinant vector shown in SEQ ID NO.4, synthesizing an insert gene shown in SEQ ID NO.5, and cloning the insert gene into the recombinant vector.

More preferably, in step (2.1), the SFV-helper plasmid is prepared by the following method:

constructing a recombinant vector, wherein the sequence of the constructed recombinant vector is SEQ ID NO. 4;

synthesizing an insert gene, wherein the sequence of the insert is SEQ ID NO. 5;

carrying out homologous recombination on the recombinant vector and the insert fragment, transforming a recombinant product into DH5 alpha competent bacteria, coating a flat plate, carrying out inverted culture, and extracting a plasmid to obtain an SFV-helper plasmid, wherein the sequence of the SFV-helper plasmid is SEQ ID NO. 6.

The invention adopts Semliki Forest Virus (SFV) as a vector to construct a packaging system, wherein one RNA molecule is an RNA replicon vector containing an inserted ASFV p32 protein. The other RNA molecule is auxiliary RNA, and after the auxiliary RNA is co-electroporated into BHK cells, the supernatant is collected to successfully prepare virus-like particles pSFV-p 32; the protein expression of p32 is successfully identified through IFA experiments, thereby providing reliable experimental basis and scientific basis for the expression screening of other important African foreign proteins and the development of mRNA vaccines, and being beneficial to the development of vaccines for preventing and controlling African swine fever epidemic diseases.

Correspondingly, the invention also provides pSFV-p32 virus-like particles, which are prepared by the preparation method. The pSFV-p32 virus-like particle can efficiently express foreign protein p32, inhibits the expression of virus structural protein and has higher biological safety.

Correspondingly, the invention also discloses application of the pSFV-p32 virus-like particle and a preparation method of the pSFV-p32 virus-like particle in RNA vaccines, therapeutic drugs and animal models. Preferably, the RNA vaccine is a vaccine for preventing and controlling African swine fever epidemic disease.

The invention is further illustrated by the following specific example

(first) preparation of the experiment

(1) Reagent

DTaqDNA polymerase, 10 XPCR BufferMgCl2(25mM), dNTP (10mM), Marker, 6 XPDNA Loading Dye, 10 XPAE, one-step method rapid competent cell preparation kit, SanPrep column type plasmid DNA small amount extraction kit, agarose, DNA column type glue recovery kit purchased from Biotechnology engineering GmbH; 4S Red Plus nucleic acid stain was purchased from BBI blood; reagents such as Fetal Bovine Serum (Fetal bone Serum), pancreatin (0.25% Trypsin-EDTA), DMEM medium (DMEM/High glucose), 0.25% Trypsin-EDTA and the like are all from Gibco company; the tissue genome DNA extraction kit is purchased from Tiangen; 2x qPCR Mix purchased from norvozam;18-T Vector, SYBR Premix ExTaqTM (Tli RNAseH plus) (RR420A), Primescript RT reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A), Gel recovery Kit (Gel Extraction Kit), Stbl3 competent cells, BamHI/AscI restriction enzymes from Takara Bio Inc. (Dalian); RNAprep pure Tissue Kit (DP431) from TIANGEN; plasmid extraction Kit (Plasmid Mini Kit)From OMEGA company; transfection reagent Lipofectamine 3000Regeant was purchased from Invitrogen; pSFVCs-lacz (#92076) was purchased from addendum; EGFP-C1 (No. 9), pSFV-sp6-EGFP plasmid (the plasmid is from invention patent of application No. 2021108223525, pSFVCs-EGFP virus-like particle and preparation method thereof) SFV-helper (the plasmid is from invention patent of application No. 2021108224072, SFV-helper plasmid).

(2) Instrument for measuring the position of a moving object

Biosafety cabinet 1300SERIES A2(Thermo corporation); a constant temperature water bath DK-8D (constant/Shanghai); carbon dioxide incubator Forma 371(Thermo corporation); biochemical incubator DHP-9162 (monogamy/Shanghai); high performance high speed table refrigerated centrifuge 5804R (Eppendorf/germany); electric heating constant temperature incubator DHP-9162 (Yiheng/Shanghai); vortex oscillator Vortex (Thermo corporation); pipette Research plus (Eppendorf/Germany); inverted microscope DMI1 (german come card); countstar cytometer IC-1000 (Shanghai Rui Yu Biotech Co., Ltd.); fluorescent quantitative PCR instrument 7500 (ABI); MicroAmp Optical 96-Well Reaction plant N8010560 (ABI); MicroAmpTM Optical additive Film4311971 (ABI); clean bench SW-CJ-2FD (Sujingtai); fluorescence microscopy (Leica, germany); CO2 incubator HF240 (likang); centrifuge NeoFuge 1600R (likang); FC500 flow cytometer (Becton-Dickinson, usa); high speed centrifuge 5840R (Eppendorf corporation, germany); ultra pure water instruments (Millipore, france); DYY-6C type current-stabilizing voltage-stabilizing electrophoresis apparatus (six one in Beijing); h6-1 mini electrophoresis tank (Shanghai Jingyi organic glass products apparatus factory); FR980 gel imaging system (shanghai complex science ltd); SMA4000 microphotometer (merinton); PCR reaction amplification apparatus (BIO Co.).

(II) contents of the experiment

(1) Constructing and obtaining pSFV-sp6-p32 plasmid;

(1.1) synthesizing an ASFV p32 gene according to the gene sequence of ASFV p32 logged in GenBank, wherein the sequence of the ASFV p32 gene is SEQ ID NO. 1;

(1.2) designing primers tongyong-F and SP6-p32-R by adopting a homologous recombination technology according to SnapGene software and synthesizing, wherein the sequence of the primer tongyong-F is SEQ ID NO.2, and the sequence of the primer SP6-p32-R is SEQ ID NO. 3;

TABLE 1 bacteria liquid identification primer

(1.3) carrying out enzyme digestion on the double-enzyme digestion plasmid pSFV-sp6-EGFP by using BamHI/SmaI;

the double-enzyme cutting plasmid pSFV-sp6-EGFP is cut by BamHI/SmaI, the cutting system is 300uL, and the cutting system is divided into 6 tubes, each tube is 50uL, and the system is shown in the following table 2:

TABLE 2 enzyme digestion System

After enzyme digestion, carrying out 1% nucleic acid gel electrophoresis identification, cutting off the gel of the band about 11045bp with obvious target bands appearing at about 726bp and 11045bp under ultraviolet light, ensuring that the size of the cut gel just contains all the target bands as much as possible, not to be too large, quickly cutting off the ultraviolet light after cutting off the gel, and preventing the target fragments from being degraded due to the over irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

In step (1.3), pSFV-sp6-EGFP was prepared by the following method:

designing and synthesizing primers, wherein the primers comprise design primers Cs-EGFP-F and Cs-EGFP-R, and bacterial liquid identification primers EGFP-identification-F and EGFP-identification-R:

amplifying a Cs-EGFP fragment by taking EGFP-C1 as a template and Cs-EGFP-F and Cs-EGFP-R as primers, and then constructing a replicon plasmid pSFVCs-sp6-EGFP through enzyme digestion, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing;

and thenpSFVCs-sp6-EGFP is taken as a template, designed and synthesized primers EGFP-F and EGFP-R are used for amplifying an EGFP fragment, and then pSFVCs-sp6-EGFP is subjected to enzyme digestion, homologous recombination, transformation and identification of a recombination product, plasmid extraction, enzyme digestion identification and sequencingAnd finally constructing a replicon plasmid pSFV-sp 6-EGFP.

The following preferred embodiments further illustrate the preparation of the pSFV-sp6-EGFP plasmid

(A) Designing and synthesizing a primer:

the primer comprises a design primer Cs-EGFP-F, Cs-EGFP-R, EGFP-F, EGFP-R and a bacterial liquid identification primer EGFP-identification-F, EGFP-identification-R, tongyong-F:

specifically, according to SnapGene software, a homologous recombination technology is adopted to design a primer Cs-EGFP-F, Cs-EGFP-R and a bacterial liquid identification primer EGFP-identification-F, EGFP-identification-R, and the primers are synthesized in Jinwei science and technology Co., Ltd (Suzhou), and are designed as the following table 1-1:

TABLE 1-1 Gene Synthesis primers

(B) Construction of replicon plasmid pSFVCS-sp 6-EGFP:

and amplifying the Cs-EGFP fragment by taking the EGFP-C1 as a template and the Cs-EGFP-F and Cs-EGFP-R as primers, and then constructing and obtaining the replicon plasmid pSFVCs-sp6-EGFP through enzyme digestion, homologous recombination, transformation and identification of a recombinant product, plasmid extraction, enzyme digestion identification and sequencing.

Wherein step (B) comprises:

(B.1) amplifying a Cs-EGFP fragment by taking the EGFP-C1 as a template and the Cs-EGFP-F and the Cs-EGFP-R as primers;

(B.2) digesting the plasmid pSFVCS-LacZ with BamHI to obtain a large fragment of the digested vector;

(B.3) carrying out homologous recombination on the Cs-EGFP fragment and the large fragment of the enzyme digestion vector to obtain a recombinant product;

(B.4) adding the recombinant product into a competent cell DH5 alpha cell to carry out recombinant product transformation;

(B.5) carrying out recombinant product identification on the transformed recombinant product;

(B.6) extracting plasmids from the identified recombinant products;

(B.7) carrying out enzyme cutting identification and sequencing on the plasmid.

Specifically, as a preferred embodiment of the present invention, the step (2) includes:

(B.1) amplification of the fragment of interest

The method is characterized in that EGFP-C1 is used as a template (figure 8), Cs-EGFP-F and Cs-EGFP-R are used as primers to amplify a Cs-EGFP fragment, and PCR systems and conditions are shown in the following tables 1-2 and tables 1-3:

TABLE 1-2 Cs-EGFP amplification PCR System

TABLE 1-3 Cs-EGFP amplification PCR program

After PCR amplification is finished, 1% nucleic acid gel electrophoresis identification is carried out, an obvious target band appears around 754bp, the gel containing the target band is cut under ultraviolet light, the size of the cut gel just contains all the target bands to the greatest extent, the gel is not too large, the ultraviolet light is cut off immediately after the gel is cut, and the target fragment is prevented from being degraded due to over-irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(B.2) BamHI digestion of plasmid pSFVCS-LacZ

Plasmid pSFVCS-LacZ (from addge) was digested with BamHI (FIG. 9) in a total of 300uL into 6 tubes of 50uL each, as shown in tables 1-4 below:

TABLE 1-4 enzyme digestion System

And (3) carrying out 0.8% nucleic acid gel electrophoresis identification after enzyme digestion, cutting the gel with a band of about 11851bp which has obvious target bands at about 3072bp and 11851bp under ultraviolet light, ensuring that the cut gel size just contains all the target bands as much as possible, and is not too large, quickly cutting the gel, immediately closing ultraviolet rays after cutting, and preventing the target fragments from being degraded due to over irradiation of the ultraviolet rays. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(B.3) homologous recombination of the target fragment and the Large fragment of the digestion vector

After the gel recovery, the target fragment and the vector fragment were prepared into the following reaction systems (tables 1 to 5) on ice according to appropriate volumes, gently shaken well, incubated in a 37 ℃ water bath for 30min, and then cooled at 4 ℃ or on ice.

TABLE 1-5 homologous recombination systems

(B.4) transformation of recombinant product

Chemically competent cells DH 5. alpha. cells for cloning were thawed on ice, 10. mu.L of the recombinant product was added to 100. mu.L of the competent cells, gently flicked against the vessel wall (Do not shake well) and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. 900 μ LSOC was added and the bacteria were shaken at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.L of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h.

(B.5) identification of the recombinant product

After overnight culture, colonies of the plates were examined and selected for bacterial liquid identification, and cultured in a shaker at 37 ℃ for about 5 hours in a 2mL EP tube containing a chloramphenicol-resistant LB medium, followed by bacterial liquid identification with an identification fragment of 413bp, and PCR systems were identified as shown in the following tables 1 to 6:

TABLE 1-6 bacteria liquid identification PCR system

And (4) taking 100uL of the bacteria liquid which is identified to be positive, adding the bacteria liquid into a 50mL centrifuge tube with the LP culture medium containing chloramphenicol resistance, shaking greatly, and culturing overnight.

(B.6) extraction of plasmid

1. Column equilibration step: 500. mu.L of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to remove the waste solution from the collection tube, and the adsorption column was replaced in the collection tube.

2. 15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible.

3. To the tube containing the pellet was added 500. mu.L of solution P1, and the pellet was suspended thoroughly using a pipette or vortex shaker.

4. The cells were lysed by adding 500. mu.L of solution P2 to the tube and gently inverting the tube 6 to 8 times. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

5. Add 700. mu.L of solution P3 to the centrifuge tube, gently turn up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

6. Transferring the supernatant collected in the previous step to an adsorption column CP3 by using a pipette, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring the waste liquid in the collection tube, and placing the adsorption column CP3 in the collection tube.

7. 600 μ L of the rinsing solution PW (please check whether absolute ethanol has been added or not) was added to the adsorption column CP3, centrifuged at 12,000rpm (-13,400 Xg) for 30-60sec, the waste liquid in the collection tube was decanted, and the adsorption column CP3 was placed in the collection tube.

8. Operation 7 is repeated.

9. The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

10. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.L of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

(B.7) enzyme cutting identification and sequencing:

the plasmid was digested with SmaI and BamHI in a total of 20uL, as shown in tables 1-7 below:

TABLE 1-7 restriction systems

And (3) carrying out 1% nucleic acid gel electrophoresis identification after enzyme digestion is finished, and sending the strip to a company Limited in Biotechnology engineering (Shanghai) for sequencing after the strip is identified correctly.

(C) Construction of replicon plasmid pSFV-sp 6-EGFP:

(C.1) amplification of the fragment of interest

To be provided withpSFVCs-sp6-EGFP is taken as a template (figure 10), EGFP-F and EGFP-R are taken as primer amplification fragments, and a PCR system and conditions are as follows:

TABLE 1-8 EGFP fragment amplification PCR System

TABLE 1-9 EGFP fragment amplification PCR program

After PCR amplification is finished, 1% nucleic acid gel electrophoresis identification is carried out, an obvious target band appears at about 745bp, the gel containing the target band is cut under ultraviolet light, the size of the cut gel just contains all the target bands as much as possible, the gel is not too large, the ultraviolet light is cut off immediately after the gel is cut, and the target fragment is prevented from being degraded due to over irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(C.2) BamHI/Bg1II enzyme digestion of double-restriction enzyme plasmidpSFVCs-sp6-EGFP

BamHI/Bg1II enzyme digestion of double digestion plasmidpSFVCs-sp6-EGFP, enzyme cutting system totally 300uL, divided into 6 tubes, each tube 50uL, system as following table 1-10:

TABLE 1-10 enzyme digestion systems

After the enzyme digestion is finished, 1% nucleic acid gel electrophoresis identification is carried out, obvious target bands appear around 1508bp and 11063bp, the gel of the bands around 11063bp is cut under ultraviolet light, the size of the cut gel is enabled to contain all the target bands as soon as possible, the size is not too large, the ultraviolet rays are cut off immediately after the gel is cut, and the target fragments are prevented from being degraded due to the over irradiation of the ultraviolet rays. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(C.3) homologous recombination of target fragment and large fragment of enzyme digestion vector

After the gel recovery, the target fragment and the vector fragment were prepared into the following reaction systems (tables 1 to 11) on ice according to appropriate volumes, gently shaken well, incubated in a 37 ℃ water bath for 30min, and then cooled at 4 ℃ or on ice.

TABLE 1-11 homologous recombination systems

(C.4) transformation of recombinant product

Chemically competent cells DH 5. alpha. cells for cloning were thawed on ice, 10. mu.L of the recombinant product was added to 100. mu.L of the competent cells, gently flicked against the vessel wall (Do not shake well) and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. Adding 900 μ L SOC, shaking the bacteria at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.L of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h.

(C.5) identification of recombinant products

After overnight culture, colonies of the plates were examined and selected for bacterial fluid identification, and cultured in a shaker at 37 ℃ for about 5 hours in a 2mL EP tube containing a chloramphenicol-resistant LP medium, followed by bacterial fluid identification with an identification fragment of 1467bp, and PCR systems were identified as shown in the following tables 1 to 12:

TABLE 1-12 bacteria liquid identification PCR system

And (4) taking 100uL of the bacteria liquid which is identified to be positive, adding the bacteria liquid into a 50mL centrifuge tube with the LP culture medium containing chloramphenicol resistance, shaking greatly, and culturing overnight.

(C.6) extraction of plasmid

1. Column equilibration step: 500. mu.L of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to remove the waste solution from the collection tube, and the adsorption column was replaced in the collection tube.

2. 15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible.

3. To the tube containing the pellet was added 500. mu.L of solution P1, and the pellet was suspended thoroughly using a pipette or vortex shaker.

4. The cells were lysed by adding 500. mu.L of solution P2 to the tube and gently inverting the tube 6 to 8 times. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

5. Add 700. mu.L of solution P3 to the centrifuge tube, gently turn up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

6. Transferring the supernatant collected in the previous step to an adsorption column CP3 by using a pipette, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring the waste liquid in the collection tube, and placing the adsorption column CP3 in the collection tube.

7. 600 μ L of the rinsing solution PW (please check whether absolute ethanol has been added or not) was added to the adsorption column CP3, centrifuged at 12,000rpm (-13,400 Xg) for 30-60sec, the waste liquid in the collection tube was decanted, and the adsorption column CP3 was placed in the collection tube.

8. Operation 7 is repeated.

9. The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

10. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.L of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

(C.7) enzyme digestion identification and sequencing:

as shown in the following tables 1-13, SmaI and BamHI were used for double digestion in a total of 20uL

TABLE 1-13 restriction systems

And carrying out 1% nucleic acid gel electrophoresis identification after enzyme digestion. After the bands are identified correctly, the bands are sent to the company of Biotechnology engineering (Shanghai) GmbH for sequencing.

Thus, a pSFV-sp6-EGFP plasmid was prepared.

(1.4) carrying out homologous recombination on the ASFV p32 gene fragment synthesized by the gene and the enzyme-cut vector fragment obtained in the step (1.3) to obtain a recombinant product;

the following reaction system was prepared on ice by mixing the gene synthesized p32 fragment and the vector fragment in appropriate volumes, as shown in Table 3 below:

TABLE 3 homologous recombination System

After gentle and gentle shaking, the mixture is incubated in a water bath kettle at 37 ℃ for 30min and then placed at 4 ℃ or cooled on ice to obtain the recombinant plasmid pSFV-sp6-p32 shown in figure 1.

(1.5) adding the recombinant product to a competent cell DH5 alpha cell to perform recombinant product transformation;

chemically competent cells for cloning, DH 5. alpha. cells, were thawed on ice, 10. mu.l of the recombinant product was added to 100. mu.l of the competent cells, gently flicked against the vessel wall (Do not shake well), and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. Adding 900 μ l SOC, shaking the bacteria at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.l of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h.

(1.6) carrying out recombinant product identification on the transformed recombinant product;

after overnight culture, colonies of the plates were examined and selected for bacterial liquid identification, and cultured in a shaker at 37 ℃ for about 5 hours in a 2mL EP tube containing a chloramphenicol-resistant LB medium, followed by bacterial liquid identification with an identification fragment of 1344bp, and PCR systems were identified as shown in the following Table 4:

TABLE 4 bacteria liquid identification PCR System

Taking 100uL of the bacteria liquid with positive identification, and carrying out large shaking and overnight culture in a 50mL centrifuge tube added with LB culture medium with chloramphenicol resistance.

(1.7) extracting the plasmid from the identified recombinant product, comprising the following steps:

1. column equilibration step: 500. mu.l of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, the waste solution in the collection tube was decanted, and the adsorption column was replaced in the collection tube.

2. 15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible.

3. To the tube containing the pellet was added 500. mu.l of solution P1, and the pellet was suspended thoroughly using a pipette or vortex shaker.

4. The cells were lysed by adding 500. mu.l of solution P2 to the tube and gently inverting the tube 6 to 8 times. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

5. Add 700. mu.l of solution P3 to the centrifuge tube, gently turn up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

6. Transferring the supernatant collected in the previous step to an adsorption column CP3 by using a pipette, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring the waste liquid in the collection tube, and placing the adsorption column CP3 in the collection tube.

7. Add 600. mu.l of rinsing solution PW (please check if absolute ethanol has been added) to the adsorption column CP3, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, dump the waste liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

8. Operation 7 is repeated.

9. The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

10. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

(1.8) carrying out enzyme digestion identification and sequencing on the plasmid.

SmaI and BamHI are used for double digestion, and the digestion system is total 20uL, as shown in the following table 5

TABLE 5 enzyme digestion System

And (3) carrying out 1% nucleic acid gel electrophoresis identification after enzyme digestion is finished, and sending the strip to a company Limited in Biotechnology engineering (Shanghai) for sequencing after the strip is identified correctly.

(2) Replicon plasmid pSFV-sp6-p32 and helper plasmid SFV-helper were transcribed in vitro;

(2.1) digesting pSFV-sp6-p32 and SFV-helper with SpeI respectively to linearize pSFV-sp6-p32 and SFV-helper;

in step (2.1), the SFV-helper plasmid is prepared by the following method:

(2.1.1) constructing a recombinant vector, wherein the sequence of the constructed recombinant vector is SEQ ID NO. 4;

(2.1.2) constructing and synthesizing an insert gene, wherein the sequence of the insert is SEQ ID NO. 5;

(2.1.3) carrying out homologous recombination on the recombinant vector and the insert, transforming a recombinant product into DH5 alpha competent bacteria, coating a flat plate, carrying out inverted culture, and extracting a plasmid to obtain an SFV-helper plasmid, wherein the sequence of the SFV-helper plasmid is SEQ ID NO. 6.

The preferred embodiments of the preparation of the SFV-helper plasmid are further illustrated below

(2.1.1) construction of recombinant vector:

(A) primer design and Synthesis

Primers were designed according to SnapGene software using homologous recombination techniques, first-F, first-R, and second-F, second-R, and synthesized in Kingwei technology Inc. (Suzhou), and the primers were designed as follows:

TABLE 2-1 primers for amplifying vector fragments

(B) Amplification of vector fragments

Amplifying fragment 1 by taking pSFVCs-lacZ (#92076) purchased from addrene as a template and taking a first segment-F and a first segment-R as primers respectively; and amplifying the fragment 2 by using the second segment-F and the second segment-R as primers, wherein the PCR system and conditions are respectively shown in the following tables 2-2, 2-3, 2-4 and 2-5:

TABLE 2-2 fragment 1 amplification PCR System

TABLE 2-3 fragment 1 amplification PCR procedure

TABLE 2-4 fragment 2 amplification PCR System

TABLE 2-5 fragment 2 amplification PCR procedure

After PCR amplification is finished, 1% nucleic acid gel electrophoresis identification is carried out, obvious target bands appear around 3042bp and 1665bp respectively, the gel containing the target bands is cut under ultraviolet light, the size of the cut gel just contains all the target bands as much as possible, the gel is not too large, the ultraviolet light is cut off immediately after the gel is cut, and the target fragments are prevented from being degraded due to over irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(C) Vector fragment 1, 2 homologous recombination

After the gel recovery, the following reaction systems were prepared on ice according to the appropriate volumes of the vector fragments 1 and 2, and the systems are as follows 2 to 6:

TABLE 2-6 homologous recombination System

Shaking gently, incubating in water bath at 37 deg.C for 30min, and cooling at 4 deg.C or on ice.

(D) Transformation of recombinant product

Chemically competent cells for cloning, DH 5. alpha. cells, were thawed on ice, 10. mu.l of the recombinant product was added to 100. mu.l of the competent cells, gently flicked against the vessel wall (Do not shake well), and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. Adding 900 μ l SOC, shaking the bacteria at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.l of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h.

(E) Identification of recombinant products

After overnight culture, colonies of the plates were examined and selected for bacterial liquid identification, and cultured in a shaker at 37 ℃ for about 5 hours in a 2mL EP tube containing a chloramphenicol-resistant LB medium, followed by bacterial liquid identification of 4691bp as an identification fragment, and PCR systems were identified as shown in the following tables 2 to 7:

TABLE 2-7 bacteria liquid identification PCR system

And (4) taking 100uL of the bacteria liquid which is identified to be positive, adding the bacteria liquid into a 50mL centrifuge tube with the LP culture medium containing chloramphenicol resistance, shaking greatly, and culturing overnight.

(F) Extraction of plasmids

Column equilibration step: 500. mu.l of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, the waste solution in the collection tube was decanted, and the adsorption column was replaced in the collection tube.

15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible.

To the tube containing the pellet was added 500. mu.l of solution P1, and the pellet was suspended thoroughly using a pipette or vortex shaker.

The cells were lysed by adding 500. mu.l of solution P2 to the tube and gently inverting the tube 6 to 8 times. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

Add 700. mu.l of solution P3 to the centrifuge tube, gently turn up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

Transferring the supernatant collected in the previous step to an adsorption column CP3 by using a pipette, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring the waste liquid in the collection tube, and placing the adsorption column CP3 in the collection tube.

Add 600. mu.l of rinsing solution PW (please check if absolute ethanol has been added) to the adsorption column CP3, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, dump the waste liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

Operation 7 is repeated.

The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

(G) Sequencing of recombinant vectors

The extracted plasmid is subjected to enzyme digestion, and after the plasmid is identified correctly, the plasmid is sent to Jinzhi Biotechnology GmbH for sequencing.

(2.1.2) Synthesis of the insert gene,

the sequence of the SFV-helper gene to be inserted is shown in SEQ ID NO.5, and the SFV-helper gene is subjected to gene synthesis.

(2.1.3) homologous recombination of the recombinant vector and the insert,

(A) homologous recombination, transformation and plasmid extraction

The recombinant vector is subjected to homologous recombination with the insert. The recombinant product was transformed into DH 5. alpha. competent bacteria, spread on ampicillin-resistant LB plates, and cultured in an inverted state at 37 ℃ for 16 hours. Shaking the colony with positive PCR, preserving the bacteria, and extracting the plasmid according to the instruction of the OMEGA plasmid miniprep kit

(B) Plasmid restriction enzyme identification and sequencing

Carrying out enzyme digestion identification on the extracted plasmid, and detecting the result by 1% agarose gel electrophoresis; the correct result is sent to Jinzhi Biotechnology GmbH for sequencing.

Thus, SFV-helper plasmids were prepared.

(2.2) in vitro transcription of linearized pSFV-sp6-p32 and SFV-helper according to mMESSAGE mMACHINE^TMIn vitro transcription was performed as described in the SP6 transcription kit (AM1340, purchased from semer plane) in the following tables 6, 7:

TABLE 6 pSFV-sp6-p32 in vitro transcription System

TABLE 7 SFV-helper in vitro transcription System

After the system is prepared, the transcription is carried out for 40min at 37 ℃, then 1uL GTP is added for continuous transcription for 2h at 37 ℃, and then 1uL TURBO DNase37 ℃ is added for 15 min. After transcription is finished, 1uL of transcription product is respectively absorbed, 4uL of nucleic-free Water and 6 × loading buffer are added for dilution, aseptic operation is paid attention to and RNA degradation is prevented, and then electrophoresis experiment is carried out in 1% SDS-page to verify whether the band is correct or not.

(2.3) co-electrotransformation of pSFV-sp6-p32 and SFV-helper in vitro transcribed mRNA into BHK cells, comprising:

1. the cells are transferred to a T75 square bottle in advance, and when the cell confluence reaches 80%, the experiment is carried out;

2. 1.5ml EP tubes and PBS were pre-cooled on ice. Precooling 0.2mm colorimetric cups (electrode cups) in a refrigerator at-20 ℃;

preheat 6ml of medium (DMEM with 1% FBS) at 3.37 ℃;

4. cells in T25 flasks were trypsinized and resuspended in 5ml complete medium;

5.500 g, centrifuging at 4 ℃ for 5 minutes;

6. the supernatant was discarded and the cell particles were washed with 10ml ice-cold PBS;

7.500 g, centrifuged at 4 ℃ for 5 minutes, and PBS washing was repeated again;

8. discarding the supernatant, completely removing residual PBS, resuspending the cells with cooled 260uL PBS, mixing uniformly, respectively sucking 260uL PBS cell suspension, and respectively placing in precooled 1.5ml EP tubes;

adding 10uL of pSFVCs-sp6-p32 and SFV-helper transcript to the PBS cell suspension respectively, and placing on ice;

10. transferring the mixture into a pre-cooled electrode cup (4mm), setting the conditions of an electric rotating instrument to be 100V and 20ms, performing electric shock once, and placing on ice for 1min after electric shock;

11. then, adding about 400ul of DMEM containing 1% FBS into the electrode cups respectively, blowing, beating and uniformly mixing, then transferring to a T25 square bottle, adding 6mL of DMEM containing 1% FBS, and placing in a 5% CO2 incubator at 37 ℃ for culture;

12. observing the cell growth condition after electroporation, collecting the supernatant after 36h, and storing at-80 ℃.

(3) And indirect immunofluorescence experiment for identifying p32 protein expression

1. The cells after collecting the supernatant are fixed overnight by methanol precooled to 4 ℃, and washed three times by PBST; normal BHK cells were used as blank control;

2. adding 5% 1mL BSA, incubating at 37 ℃ for 1h, and washing with PBST three times;

3. adding 1500-fold diluted African swine fever serum into each hole, incubating for 1h at 37 ℃, and washing for three times by PBST;

4. adding goat anti-pig IgG labeled with IFTC diluted by 200 times into each well, incubating for 1h at 37 ℃, washing PBST for three times, and taking care of keeping out of the sun;

5, the plate was observed under a fluorescence microscope and compared with normal cells.

(4) pSFV-p32 viral particle TCID₅₀Measurement of (2)

pSFV-p32 virus was serially diluted 10-fold in DMEM medium, i.e., 100. mu.L of each dilution 10-1 and 10-2 … 10-8 was added to a 96-well cell culture plate, followed by 100. mu.L of 0.25% pancreatin-digested BHK-21 cells (cell content is preferably about 105/mL), each dilution was repeated 8 times, and a blank cell culture control was set. The mixture was placed in a 37 ℃ 5% CO2 incubator. Cytopathic effects were observed day by day and the number of cytopathic wells was recorded until control cells were aged off. The TCID50 of the virus was calculated according to the Reed-Muench method.

(III) results of the experiment

(1) BamHI/SmaI enzyme digestion double-restriction enzyme digestion plasmid pSFV-sp6-EGFP

After the BamHI/SmaI enzyme digestion double-enzyme digestion plasmid pSFV-sp6-EGFP, the following picture segments are 11051bp and 725bp respectively which are consistent with theoretical values as shown in the following figure 2. In FIG. 2, M is 15000,10000,7500,5000,3000,1500,1000,500 from top to bottom, respectively, and 1 is SmaI and BamH double enzyme digestion fragment.

(2) Identification of recombinant plasmid liquid

8 colonies were picked up and identified, as shown in the following FIG. 3, the size of colonies 2,5,6,7,8 was 1344bp, which is consistent with the theoretical value. In FIG. 3, M is 2000, 1000, 750, 500, 250, 100 from top to bottom, respectively.

(3) Enzyme digestion identification and sequencing

Carrying out double enzyme digestion on the successfully identified plasmid by SmaI and BamHI, wherein the sizes of the fragments shown in figure 4 are 593bp and 11051bp respectively, and are consistent with the size of a theoretical value; sending the obtained product to a company Limited in Biotechnology engineering (Shanghai) for sequencing, wherein the sequencing results are completely the same.

In FIG. 4, M is 15000,10000,7500,5000,3000,1500,1000,500 from top to bottom; 1 is SmaI and BamH double enzyme digestion fragment.

(4) Replicon plasmid pSFV-sp6-p32 and helper plasmid SFV-helper for in vitro transcription

After digestion of pSFV-sp6-p32 and SFV-helper, respectively, with SpeI, according to mMESSAGE mMACHINE^TMIn vitro transcription was performed as described in the SP6 transcription kit (AM1340, purchased from semer fly) in fig. 5, 6:

in FIG. 5, M1 is 15000,10000,7500,5000,3000,1500,1000,500 from top to bottom; m2 is 8000,5000,3000,1500, 1000,500 from top to bottom; lane 2 shows the in vitro transcription result of pSFV-sp6-p 32;

in FIG. 6, M is 8000,5000,3000,1500, 1000,500, respectively, from top to bottom; lane 4 shows the in vitro transcription results of SFV-helper.

As can be seen from FIGS. 5 and 6, the bands of interest after electrophoresis of pSFV-sp6-p32 and SFV-helper in vitro transcripts coincided with the theoretical values.

(5) Identification of p32 protein expression by indirect immunofluorescence assay

The in vitro transcribed pSFVCs-sp6-p32 and SFV-helper cells are electrified and transferred into BHK cells, then the BHK cells are inoculated into a T25 square flask for culture, and after 36h, the cells after collecting the supernatant are subjected to IFA to identify the expression of ASFV p32 protein, and as a result, stronger fluorescence expression is found, but no fluorescence expression is found in a negative control group (figure 7).

(6) pSFV-p32 viral particle TCID₅₀Measurement of (2)

The titer of the virus was 10 according to the Reed-Muench method^-3TCID₅₀/_0.1mLThe titer meets the requirements of subsequent animal experiments.

In conclusion, the protein expression of p32 is successfully identified through IFA experiments, and reliable experimental basis and scientific basis are provided for expression screening and mRNA vaccine development of other important African foreign proteins.

The above description is only a preferred embodiment of the present invention, and certainly should not be taken as limiting the scope of the invention, which is defined by the claims and their equivalents.

Sequence listing

<110> Chongqing research institute of BioIndustrial technology, Inc

<120> pSFV-p32 virus-like particle, preparation method and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 627

<212> DNA

<213> Artificial Synthesis

<400> 1

atagcaccat gaatggatcc atggatttta ttttaaatat atccatgaaa atggaggtca 60

tcttcaaaac ggatttaaga tcatcttcac aagttgtgtt tcatgcgggt agcctgtata 120

attggttttc tgttgagatt atcaatagcg gtagaattgt tacgaccgct ataaaaacat 180

tgcttagtac tgttaagtat gatattgtga aatctgctcg tatatatgca gggcaagggt 240

atactgaaca tcaggctcaa gaagaatgga atatgattct gcatgtgctg tttgaagagg 300

agacggaatc ctcagcatct tcggagaaca ttcatgaaaa aaatgataat gaaaccaatg 360

aatgcacatc ctcctttgaa acgttgtttg agcaagagcc ctcatcggag gtacctaaag 420

actccaagct gtatatgctt gcacaaaaga ctgtgcaaca tattgaacaa tatggaaagg 480

cacctgattt taacaaggtt attagagcac ataattttat tcaaaccatt tatggaaccc 540

ctctaaagga agaagaaaaa gaggtggtaa gactcatggt tattaaactt ttaaaaaaaa 600

aataacccgg gtaattaatt gaattac 627

<210> 2

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 2

ggtttaatga tcctcgaaga tc 22

<210> 3

<211> 55

<212> DNA

<213> Artificial Synthesis

<400> 3

gtaattcaat taattacccg ggttattttt tttttaaaag tttaataacc atgag 55

<210> 4

<211> 3538

<212> DNA

<213> Artificial Synthesis

<400> 4

cactataccg acaggagcgg gcaaaccggg agacagtggc cggcccatct ttgacaacaa 60

gggtagggta gtcgctatcg tcctgggcgg ggccaacgag ggctcacgca cagcactgtc 120

ggtggtcacc tggaacaaag atatggtgac tagagtgacc cccgaggggt ccgaagagtg 180

gtccgccccg ctgattactg ccatgtgtgt ccttgccaat gctaccttcc cgtgcttcca 240

gcccccgtgt gtaccttgct gctatgaaaa caacgcagag gccacactac ggatgctcga 300

ggataacgtg gataggccag ggtactacga cctccttcag gcagccttga cgtgccgaaa 360

cggaacaaga caccggcgca gcgtgtcgca acacttcaac gtgtataagg ctacacgccc 420

ttacatcgcg tactgcgccg actgcggagc agggcactcg tgtcatagcc ccgtagcaat 480

tgaagcggtc aggtccgaag ctaccgacgg gatgctgaag attcagttct cggcacaaat 540

tggcatagat aagagtgaca atcatgacta cacgaagata aggtacgcag acgggcacgc 600

cattgagaat gccgtccggt catctttgaa ggtagccacc tccggagact gtttcgtcca 660

tggcacaatg ggacatttca tactggcaaa gtgcccaccg ggtgaattcc tgcaggtctc 720

gatccaggac accagaaacg cggtccgtgc ctgcagaata caatatcatc atgaccctca 780

accggtgggt agagaaaaat ttacaattag accacactat ggaaaagaga tcccttgcac 840

cacttatcaa cagaccacag cgaagaccgt ggaggaaatc gacatgcata tgccgccaga 900

tacgccggac aggacgttgc tatcacagca atctggcaat gtaaagatca cagtcggagg 960

aaagaaggtg aaatacaact gcacctgtgg aaccggaaac gttggcacta ctaattcgga 1020

catgacgatc aacacgtgtc taatagagca gtgccacgtc tcagtgacgg accataagaa 1080

atggcagttc aactcacctt tcgtcccgag agccgacgaa ccggctagaa aaggcaaagt 1140

ccatatccca ttcccgttgg acaacatcac atgcagagtt ccaatggcgc gcgaaccaac 1200

cgtcatccac ggcaaaagag aagtgacact gcaccttcac ccagatcatc ccacgctctt 1260

ttcctaccgc acactgggtg aggacccgca gtatcacgag gaatgggtga cagcggcggt 1320

ggaacggacc atacccgtac cagtggacgg gatggagtac cactggggaa acaacgaccc 1380

agtgaggctt tggtctcaac tcaccactga agggaaaccg cacggctggc cgcatcagat 1440

cgtacagtac tactatgggc tttacccggc cgctacagta tccgcggtcg tcgggatgag 1500

cttactggcg ttgatatcga tcttcgcgtc gtgctacatg ctggttgcgg cccgcagtaa 1560

gtgcttgacc ccttatgctt taacaccagg agctgcagtt ccgtggacgc tggggatact 1620

ctgctgcgcc ccgcgggcgc acgcagctag tgtggcagag actatggcct acttgtggga 1680

ccaaaaccaa gcgttgttct ggttggagtt tgcggcccct gttgcctgca tcctcatcat 1740

cacgtattgc ctcagaaacg tgctgtgttg ctgtaagagc ctttcttttt tagtgctact 1800

gagcctcggg gcaaccgcca gagcttacga acattcgaca gtaatgccga acgtggtggg 1860

gttcccgtat aaggctcaca ttgaaaggcc aggatatagc cccctcactt tgcagatgca 1920

ggttgttgaa accagcctcg aaccaaccct taatttggaa tacataacct gtgagtacaa 1980

gacggtcgtc ccgtcgccgt acgtgaagtg ctgcggcgcc tcagagtgct ccactaaaga 2040

gaagcctgac taccaatgca aggtttacac aggcgtgtac ccgttcatgt ggggaggggc 2100

atattgcttc tgcgactcag aaaacacgca actcagcgag gcgtacgtcg atcgatcgga 2160

cgtatgcagg catgatcacg catctgctta caaagcccat acagcatcgc tgaaggccaa 2220

agtgagggtt atgtacggca acgtaaacca gactgtggat gtttacgtga acggagacca 2280

tgccgtcacg atagggggta ctcagttcat attcgggccg ctgtcatcgg cctggacccc 2340

gttcgacaac aagatagtcg tgtacaaaga cgaagtgttc aatcaggact tcccgccgta 2400

cggatctggg caaccagggc gcttcggcga catccaaagc agaacagtgg agagtaacga 2460

cctgtacgcg aacacggcac tgaagctggc acgcccttca cccggcatgg tccatgtacc 2520

gtacacacag acaccttcag ggttcaaata ttggctaaag gaaaaaggga cagccctaaa 2580

tacgaaggct ccttttggct gccaaatcaa aacgaaccct gtcagggcca tgaactgcgc 2640

cgtgggaaac atccctgtct ccatgaattt gcctgacagc gcctttaccc gcattgtcga 2700

ggcgccgacc atcattgacc tgacttgcac agtggctacc tgtacgcact cctcggattt 2760

cggcggcgtc ttgacactga cgtacaagac cgacaagaac ggggactgct ctgtacactc 2820

gcactctaac gtagctactc tacaggaggc cacagcaaaa gtgaagacag caggtaaggt 2880

gaccttacac ttctccacgg caagcgcatc accttctttt gtggtgtcgc tatgcagtgc 2940

tagggccacc tgttcagcgt cgtgtgagcc cccgaaagac cacatagtcc catatgcggc 3000

tagccacagt aacgtagtgt ttccagacat gtcgggcacc gcactatcat gggtgcagaa 3060

aatctcgggt ggtctggggg ccttcgcaat cggcgctatc ctggtgctgg ttgtggtcac 3120

ttgcattggg ctccgcagat aagttagggt aggcaatggc attgatatag caagaaaatt 3180

gaaaacagaa aaagttaggg taagcaatgg catataacca taactgtata acttgtaaca 3240

aagcgcaaca agacctgcgc aattggcccc gtggtccgcc tcacggaaac tcggggcaac 3300

tcatattgac acattaattg gcaataattg gaagcttaca taagcttaat tcgacgaata 3360

attggatttt tattttattt tgcaattggt ttttaatatt tccaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactagtct 3480

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttcc 3538

<210> 5

<211> 3538

<212> DNA

<213> Artificial Synthesis

<400> 5

cactataccg acaggagcgg gcaaaccggg agacagtggc cggcccatct ttgacaacaa 60

gggtagggta gtcgctatcg tcctgggcgg ggccaacgag ggctcacgca cagcactgtc 120

ggtggtcacc tggaacaaag atatggtgac tagagtgacc cccgaggggt ccgaagagtg 180

gtccgccccg ctgattactg ccatgtgtgt ccttgccaat gctaccttcc cgtgcttcca 240

gcccccgtgt gtaccttgct gctatgaaaa caacgcagag gccacactac ggatgctcga 300

ggataacgtg gataggccag ggtactacga cctccttcag gcagccttga cgtgccgaaa 360

cggaacaaga caccggcgca gcgtgtcgca acacttcaac gtgtataagg ctacacgccc 420

ttacatcgcg tactgcgccg actgcggagc agggcactcg tgtcatagcc ccgtagcaat 480

tgaagcggtc aggtccgaag ctaccgacgg gatgctgaag attcagttct cggcacaaat 540

tggcatagat aagagtgaca atcatgacta cacgaagata aggtacgcag acgggcacgc 600

cattgagaat gccgtccggt catctttgaa ggtagccacc tccggagact gtttcgtcca 660

tggcacaatg ggacatttca tactggcaaa gtgcccaccg ggtgaattcc tgcaggtctc 720

gatccaggac accagaaacg cggtccgtgc ctgcagaata caatatcatc atgaccctca 780

accggtgggt agagaaaaat ttacaattag accacactat ggaaaagaga tcccttgcac 840

cacttatcaa cagaccacag cgaagaccgt ggaggaaatc gacatgcata tgccgccaga 900

tacgccggac aggacgttgc tatcacagca atctggcaat gtaaagatca cagtcggagg 960

aaagaaggtg aaatacaact gcacctgtgg aaccggaaac gttggcacta ctaattcgga 1020

catgacgatc aacacgtgtc taatagagca gtgccacgtc tcagtgacgg accataagaa 1080

atggcagttc aactcacctt tcgtcccgag agccgacgaa ccggctagaa aaggcaaagt 1140

ccatatccca ttcccgttgg acaacatcac atgcagagtt ccaatggcgc gcgaaccaac 1200

cgtcatccac ggcaaaagag aagtgacact gcaccttcac ccagatcatc ccacgctctt 1260

ttcctaccgc acactgggtg aggacccgca gtatcacgag gaatgggtga cagcggcggt 1320

ggaacggacc atacccgtac cagtggacgg gatggagtac cactggggaa acaacgaccc 1380

agtgaggctt tggtctcaac tcaccactga agggaaaccg cacggctggc cgcatcagat 1440

cgtacagtac tactatgggc tttacccggc cgctacagta tccgcggtcg tcgggatgag 1500

cttactggcg ttgatatcga tcttcgcgtc gtgctacatg ctggttgcgg cccgcagtaa 1560

gtgcttgacc ccttatgctt taacaccagg agctgcagtt ccgtggacgc tggggatact 1620

ctgctgcgcc ccgcgggcgc acgcagctag tgtggcagag actatggcct acttgtggga 1680

ccaaaaccaa gcgttgttct ggttggagtt tgcggcccct gttgcctgca tcctcatcat 1740

cacgtattgc ctcagaaacg tgctgtgttg ctgtaagagc ctttcttttt tagtgctact 1800

gagcctcggg gcaaccgcca gagcttacga acattcgaca gtaatgccga acgtggtggg 1860

gttcccgtat aaggctcaca ttgaaaggcc aggatatagc cccctcactt tgcagatgca 1920

ggttgttgaa accagcctcg aaccaaccct taatttggaa tacataacct gtgagtacaa 1980

gacggtcgtc ccgtcgccgt acgtgaagtg ctgcggcgcc tcagagtgct ccactaaaga 2040

gaagcctgac taccaatgca aggtttacac aggcgtgtac ccgttcatgt ggggaggggc 2100

atattgcttc tgcgactcag aaaacacgca actcagcgag gcgtacgtcg atcgatcgga 2160

cgtatgcagg catgatcacg catctgctta caaagcccat acagcatcgc tgaaggccaa 2220

agtgagggtt atgtacggca acgtaaacca gactgtggat gtttacgtga acggagacca 2280

tgccgtcacg atagggggta ctcagttcat attcgggccg ctgtcatcgg cctggacccc 2340

gttcgacaac aagatagtcg tgtacaaaga cgaagtgttc aatcaggact tcccgccgta 2400

cggatctggg caaccagggc gcttcggcga catccaaagc agaacagtgg agagtaacga 2460

cctgtacgcg aacacggcac tgaagctggc acgcccttca cccggcatgg tccatgtacc 2520

gtacacacag acaccttcag ggttcaaata ttggctaaag gaaaaaggga cagccctaaa 2580

tacgaaggct ccttttggct gccaaatcaa aacgaaccct gtcagggcca tgaactgcgc 2640

cgtgggaaac atccctgtct ccatgaattt gcctgacagc gcctttaccc gcattgtcga 2700

ggcgccgacc atcattgacc tgacttgcac agtggctacc tgtacgcact cctcggattt 2760

cggcggcgtc ttgacactga cgtacaagac cgacaagaac ggggactgct ctgtacactc 2820

gcactctaac gtagctactc tacaggaggc cacagcaaaa gtgaagacag caggtaaggt 2880

gaccttacac ttctccacgg caagcgcatc accttctttt gtggtgtcgc tatgcagtgc 2940

tagggccacc tgttcagcgt cgtgtgagcc cccgaaagac cacatagtcc catatgcggc 3000

tagccacagt aacgtagtgt ttccagacat gtcgggcacc gcactatcat gggtgcagaa 3060

aatctcgggt ggtctggggg ccttcgcaat cggcgctatc ctggtgctgg ttgtggtcac 3120

ttgcattggg ctccgcagat aagttagggt aggcaatggc attgatatag caagaaaatt 3180

gaaaacagaa aaagttaggg taagcaatgg catataacca taactgtata acttgtaaca 3240

aagcgcaaca agacctgcgc aattggcccc gtggtccgcc tcacggaaac tcggggcaac 3300

tcatattgac acattaattg gcaataattg gaagcttaca taagcttaat tcgacgaata 3360

attggatttt tattttattt tgcaattggt ttttaatatt tccaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactagtct 3480

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttcc 3538

<210> 6

<211> 8192

<212> DNA

<213> Artificial Synthesis

<400> 6

atttaggtga cactatagat ggcggatgtg tgacatacac gacgccaaaa gattttgttc 60

cagctcctgc cacctccgct acgcgagaga ttaaccaccc acgatggccg ccaaagtgca 120

tgttgatatt gaggctgaca gcccattcat caagtctttg cagaaggcat ttccgtcgtt 180

cgaggtggag tcattgcagg tcacaccaaa tgaccatgca aatgccagag cattttcgca 240

cctggctacc aaattgatcg agcaggagac tgacaaagac acactcatct tggatatcgg 300

cagtgcgcct tccaggagaa tgatgtcgac atgaaacgag atgtcaaagt cactccaggg 360

acgaaacaca cagaggaaag acccaaagtc caggtaattc aagcagcgga gccattggcg 420

accgcttacc tgtgcggcat ccacagggaa ttagtaagga gactaaatgc tgtgttacgc 480

cctaacgtgc acacattgtt tgatatgtcg gccgaagact ttgacgcgat catcgcctct 540

cacttccacc caggagaccc ggttctagag acggacattg catcattcga caaaagccag 600

gacgactcct tggctcttac aggtttaatg atcctcgaag atctaggggt ggatcagtac 660

ctgctggact tgatcgaggc agcctttggg gaaatatcca gctgtcacct accaactggc 720

acgcgcttca agttcggagc tatgatgaaa tcgggcatgt ttctgacttt gtttattaac 780

actgttttga acatcaccat agcaagcagg gtactggagc agagactcac tgactccgcc 840

tgtgcggcct tcatcggcga cgacaacatc gttcacggag tgatctccga caagctgatg 900

gcggagaggt gcgcgtcgtg ggtcaacatg gaggtgaaga tcattgacgc tgtcatgggc 960

gaaaaacccc catatttttg tgggggattc atagtttttg acagcgtcac acagaccgcc 1020

tgccgtgttt cagacccact taagcgcctg ttcaagttgg gtaagccgct aacagctgaa 1080

gacaagcagg acgaagacag gcgacgagca ctgagtgacg aggttagcaa gtggttccgg 1140

acaggcttgg gggccgaact ggaggtggca ctaacatcta ggtatgaggt agagggctgc 1200

aaaagtatcc tcatagccat ggccaccttg gcgagggaca ttaaggcgtt taagaaattg 1260

agaggacctg ttatacacct ctacggcggt cctagattgg tgcgttaata cacagaattc 1320

tgattatagc gcactattat agcaccatga attacatccc tacgcaaacg ttttacggcc 1380

gccggtggcg cccgcgcccg gcggcccgtc cctggccgtt gcaggccact ccggtggctc 1440

ccgtcgtccc cgacttccag gcccagcaga tgcagcaact catcagcgcc gtaaatgcgc 1500

tgacaatgag acagaacgca attgctcctg ctaggcctcc caaaccaaag aagaagaaga 1560

caaccaaacc aaagccgaaa acgcagccca agaagatcaa cggaaaaacg cagcagcaaa 1620

agaagaaaga caagcaagcc gacaagaaga agaagaaacc cggaaaaaga gaaagaatgt 1680

gcatgaagat tgaaaatgac tgtatcttcg aagtcaaaca cgaaggaaag gtcactgggt 1740

acgcctgcct ggtgggcgac aaagtcatga aacctgccca cgtgaaagga gtcatcgaca 1800

acgcggacct ggcaaagcta gctttcaaga aatcgagcaa gtatgacctt gagtgtgccc 1860

agataccagt tcacatgagg tcggatgcct caaagtacac gcatgagaag cccgagggac 1920

actataactg gcaccacggg gctgttcagt acagcggagg taggttcact ataccgacag 1980

gagcgggcaa accgggagac agtggccggc ccatctttga caacaagggt agggtagtcg 2040

ctatcgtcct gggcggggcc aacgagggct cacgcacagc actgtcggtg gtcacctgga 2100

acaaagatat ggtgactaga gtgacccccg aggggtccga agagtggtcc gccccgctga 2160

ttactgccat gtgtgtcctt gccaatgcta ccttcccgtg cttccagccc ccgtgtgtac 2220

cttgctgcta tgaaaacaac gcagaggcca cactacggat gctcgaggat aacgtggata 2280

ggccagggta ctacgacctc cttcaggcag ccttgacgtg ccgaaacgga acaagacacc 2340

ggcgcagcgt gtcgcaacac ttcaacgtgt ataaggctac acgcccttac atcgcgtact 2400

gcgccgactg cggagcaggg cactcgtgtc atagccccgt agcaattgaa gcggtcaggt 2460

ccgaagctac cgacgggatg ctgaagattc agttctcggc acaaattggc atagataaga 2520

gtgacaatca tgactacacg aagataaggt acgcagacgg gcacgccatt gagaatgccg 2580

tccggtcatc tttgaaggta gccacctccg gagactgttt cgtccatggc acaatgggac 2640

atttcatact ggcaaagtgc ccaccgggtg aattcctgca ggtctcgatc caggacacca 2700

gaaacgcggt ccgtgcctgc agaatacaat atcatcatga ccctcaaccg gtgggtagag 2760

aaaaatttac aattagacca cactatggaa aagagatccc ttgcaccact tatcaacaga 2820

ccacagcgaa gaccgtggag gaaatcgaca tgcatatgcc gccagatacg ccggacagga 2880

cgttgctatc acagcaatct ggcaatgtaa agatcacagt cggaggaaag aaggtgaaat 2940

acaactgcac ctgtggaacc ggaaacgttg gcactactaa ttcggacatg acgatcaaca 3000

cgtgtctaat agagcagtgc cacgtctcag tgacggacca taagaaatgg cagttcaact 3060

cacctttcgt cccgagagcc gacgaaccgg ctagaaaagg caaagtccat atcccattcc 3120

cgttggacaa catcacatgc agagttccaa tggcgcgcga accaaccgtc atccacggca 3180

aaagagaagt gacactgcac cttcacccag atcatcccac gctcttttcc taccgcacac 3240

tgggtgagga cccgcagtat cacgaggaat gggtgacagc ggcggtggaa cggaccatac 3300

ccgtaccagt ggacgggatg gagtaccact ggggaaacaa cgacccagtg aggctttggt 3360

ctcaactcac cactgaaggg aaaccgcacg gctggccgca tcagatcgta cagtactact 3420

atgggcttta cccggccgct acagtatccg cggtcgtcgg gatgagctta ctggcgttga 3480

tatcgatctt cgcgtcgtgc tacatgctgg ttgcggcccg cagtaagtgc ttgacccctt 3540

atgctttaac accaggagct gcagttccgt ggacgctggg gatactctgc tgcgccccgc 3600

gggcgcacgc agctagtgtg gcagagacta tggcctactt gtgggaccaa aaccaagcgt 3660

tgttctggtt ggagtttgcg gcccctgttg cctgcatcct catcatcacg tattgcctca 3720

gaaacgtgct gtgttgctgt aagagccttt cttttttagt gctactgagc ctcggggcaa 3780

ccgccagagc ttacgaacat tcgacagtaa tgccgaacgt ggtggggttc ccgtataagg 3840

ctcacattga aaggccagga tatagccccc tcactttgca gatgcaggtt gttgaaacca 3900

gcctcgaacc aacccttaat ttggaataca taacctgtga gtacaagacg gtcgtcccgt 3960

cgccgtacgt gaagtgctgc ggcgcctcag agtgctccac taaagagaag cctgactacc 4020

aatgcaaggt ttacacaggc gtgtacccgt tcatgtgggg aggggcatat tgcttctgcg 4080

actcagaaaa cacgcaactc agcgaggcgt acgtcgatcg atcggacgta tgcaggcatg 4140

atcacgcatc tgcttacaaa gcccatacag catcgctgaa ggccaaagtg agggttatgt 4200

acggcaacgt aaaccagact gtggatgttt acgtgaacgg agaccatgcc gtcacgatag 4260

ggggtactca gttcatattc gggccgctgt catcggcctg gaccccgttc gacaacaaga 4320

tagtcgtgta caaagacgaa gtgttcaatc aggacttccc gccgtacgga tctgggcaac 4380

cagggcgctt cggcgacatc caaagcagaa cagtggagag taacgacctg tacgcgaaca 4440

cggcactgaa gctggcacgc ccttcacccg gcatggtcca tgtaccgtac acacagacac 4500

cttcagggtt caaatattgg ctaaaggaaa aagggacagc cctaaatacg aaggctcctt 4560

ttggctgcca aatcaaaacg aaccctgtca gggccatgaa ctgcgccgtg ggaaacatcc 4620

ctgtctccat gaatttgcct gacagcgcct ttacccgcat tgtcgaggcg ccgaccatca 4680

ttgacctgac ttgcacagtg gctacctgta cgcactcctc ggatttcggc ggcgtcttga 4740

cactgacgta caagaccgac aagaacgggg actgctctgt acactcgcac tctaacgtag 4800

ctactctaca ggaggccaca gcaaaagtga agacagcagg taaggtgacc ttacacttct 4860

ccacggcaag cgcatcacct tcttttgtgg tgtcgctatg cagtgctagg gccacctgtt 4920

cagcgtcgtg tgagcccccg aaagaccaca tagtcccata tgcggctagc cacagtaacg 4980

tagtgtttcc agacatgtcg ggcaccgcac tatcatgggt gcagaaaatc tcgggtggtc 5040

tgggggcctt cgcaatcggc gctatcctgg tgctggttgt ggtcacttgc attgggctcc 5100

gcagataagt tagggtaggc aatggcattg atatagcaag aaaattgaaa acagaaaaag 5160

ttagggtaag caatggcata taaccataac tgtataactt gtaacaaagc gcaacaagac 5220

ctgcgcaatt ggccccgtgg tccgcctcac ggaaactcgg ggcaactcat attgacacat 5280

taattggcaa taattggaag cttacataag cttaattcga cgaataattg gatttttatt 5340

ttattttgca attggttttt aatatttcca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tagtctgcat taatgaatcg 5460

gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 5520

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5580

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5640

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5700

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5760

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5820

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5880

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5940

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6000

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6060

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6120

gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6180

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6240

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6300

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 6360

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 6420

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 6480

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 6540

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 6600

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 6660

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 6720

cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 6780

cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 6840

ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 6900

tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 6960

catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7020

gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 7080

gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7140

tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7200

catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7260

aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 7320

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 7380

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7440

aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7500

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 7560

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 7620

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 7680

accattcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg 7740

aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt 7800

cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 7860

tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 7920

gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatctggct agcgatgacc 7980

ctgctgattg gttcgctgac catttccggg tgcgggacgg cgttaccaga aactcagaag 8040

gttcgtccaa ccaaaccgac tctgacggca gtttacgaga gagatgatag ggtctgcttc 8100

agtaagccag atgctacaca attaggcttg tacatattgt cgttagaacg cggctacaat 8160

taatacataa ccttatgtat catacacata cg 8192

<210> 7

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 7

gtattgggcg ctcttccgct tcctcgctca c 31

<210> 8

<211> 32

<212> DNA

<213> Artificial Synthesis

<400> 8

gtttcatgtc gacatcattc tcctggaagg cg 32

<210> 9

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 9

gatgtcgaca tgaaacgaga tgtcaaagtc 30

<210> 10

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 10

ccgctcctgt cggtatagtg aacctacctc 30

<210> 11

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 11

gtccgaagag tgggatccca tggtgagcaa g 31

<210> 12

<211> 33

<212> DNA

<213> Artificial Synthesis

<400> 12

ttcaattaat tacccgggct tgtacagctc gtc 33

<210> 13

<211> 17

<212> DNA

<213> Artificial Synthesis

<400> 13

ccggcaagct gcccgtg 17

<210> 14

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 14

gggggtgttc tgctggtag 19

<210> 15

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 15

ggtttaatga tcctcgaaga tc 22

<210> 16

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 16

ccttgctcac catgggatcc ggtgctataa tagtg 35

<210> 17

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 17

ggtttaatga tcctcgaaga tc 22

<210> 18

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 18

ccttgctcac catgggatcc ggtgctataa tagtg 35