Circovirus type 3 double-copy full-length gene infectious clone plasmid and construction method and application thereof

文档序号：1871891 发布日期：2021-11-23 浏览：3次中文

阅读说明：本技术 一种圆圈病毒3型双拷贝全长基因感染性克隆质粒及其构建方法和应用 (Circovirus type 3 double-copy full-length gene infectious clone plasmid and construction method and application thereof ) 是由成子强张贤文于 2021-08-16 设计创作，主要内容包括：本发明涉及病毒学和生物技术领域,涉及一种利用反向遗传学技术的圆圈病毒3型(GyV3)双拷贝全长基因感染性克隆及其构建方法和应用,具体利用GyV3的环状基因组结构特点和自带的酶切位点,使用高保真酶扩增到两段不同起止点的病毒全基因组,通过两次连接转化连接于真核表达pcDNA3.1(+)构建GyV3双拷贝全长感染性克隆pcDNA3.1-2GyV3,并且实现SPF鸡体内的病毒拯救和致病性研究；最终提供了一种简单、快捷的圆圈病毒反向遗传学操作系统,为GyV3致病性及基因结构与功能研究等方面提供平台,也为研究针对GyV3的基因工程疫苗奠定了基础。(The invention relates to the field of virology and biotechnology, and relates to a circovirus type 3 (GyV3) double-copy full-length gene infectious clone using a reverse genetics technology, a construction method and application thereof, in particular to a method for amplifying two sections of virus whole genomes with different starting and stopping points by using GyV3 circular genome structural characteristics and self-contained enzyme cutting sites, connecting the virus whole genomes with eukaryotic expression pcDNA3.1(+) through two times of connection transformation to construct GyV3 double-copy full-length infectious clone pcDNA3.1-2GyV3, and realizing virus rescue and pathogenicity research in SPF chickens; finally, a simple and quick circovirus reverse genetics operating system is provided, a platform is provided for GyV3 pathogenicity and gene structure and function research, and a foundation is laid for researching a GyV3 genetic engineering vaccine.)

1. A novel chicken circovirus type 3 strain is characterized in that: is named as GyV3 SDAU-2 strain, and the biological preservation number is CCTCC NO: V202061.

2. A circovirus type 3 double-copy whole genome infectious clonal plasmid, characterized in that: the cloning plasmid consists of a eukaryotic expression vector pcDNA3.1(+) and a circovirus type 3 double-copy whole genome, and is named as pcDNA3.1-2GyV3, wherein the nucleotide sequence of double-copy whole genome infectious cloning is shown as SEQ ID NO. 1.

3. The use of a circovirus type 3 double-copy whole genome infectious clonal plasmid of claim 1, wherein: the plasmid is used for virus rescue and pathogenicity research in circovirus type 3 bodies.

Technical Field

The invention relates to the field of virology and biotechnology, and relates to a circovirus type 3 double-copy full-length gene infectious clone by utilizing reverse genetics technology, and a construction method and application thereof.

Background

Circovirus type 3 (Gyrovirus 3, GyV3), the third circovirus member following the Chicken infectious anemia virus (Chicken infectious anaemia virus, CAV or CIAV), is classified in the family dactyloviridae (angioviridae) of the single-stranded Circular DNA virus phylum (Circular replication-associated protein-encoding single-stranded DNA viruses) that encodes replication-related proteins. GyV3 since the name was named by macroviromics identification in acute gastroenteritis diarrhea feces of unknown etiology of chile children in 2012, it was found in diarrhea feces of different ages of human, ferret diarrhea feces and chicken in market. GyV3 has attracted common interest in the medical and veterinary community because of its broad and complex nature of finding samples, its relevance to disease and host range. In 2018, the inventor identifies GyV3 in the glandular stomach of white feather broiler clinically with proventriculitis of unknown etiology, and finds that the infection rate of the glandular stomach of the broiler is 12.5% (42/336) in the glandular gastritis disease material collected in 2013 and 2017 in retrospective epidemiological investigation, and the infection rate can infect chickens and mice clearly in the subsequent pathogenicity experiment, and meanwhile, horizontal transmission between two groups is realized. Therefore, GyV3 has great relevance with broiler proventriculitis, and the cross-species transmission phenomenon indicates potential public health significance, and deep research is urgently needed, but due to the lack of mature separation and purification technology and an effective in vitro culture system, the exact pathogenicity and research of GyV3 are seriously hindered.

GyV3 the gene structure is a single-stranded circular negative-sense DNA of 2.3Kb in size, comprising 3 homologously overlapping Open Reading Frames (ORFs) and a noncoding region (UTR). The 3 ORFs encode 3 viral proteins, capsid protein VP1, scaffold protein VP2, and apoptosis protein VP3, respectively, which are translated from a single polycistronic mRNA by alternating the initiation codon, but the true mechanism of codon usage has not been elucidated. The replication pattern of circovirus has not been acknowledged by all scientists and to date there is a lack of definitive conclusions. The mainstream view is that its DNA is replicated by Rolling Circle Replication (RCR), but it is well established that viruses lack the mechanism for replicating their own DNA, rely on dividing host cells for DNA replication, and undergo a double-stranded DNA replication form (dsRF) in the process, which lays the molecular foundation for the general PCR amplification and infectious clone construction of GyV3 whole genomes.

Infectious clone is used as an important research tool in virus reverse genetics research, and is widely applied to rescue of RNA virus and other gene type viruses, and basic and application research such as virus gene structure and function, virus pathogenicity, virus host interaction, vaccine development and the like. Infectious clones refer to artificially constructed genetic material that can achieve infection in vitro and in vivo and assemble infectious virions. GyV3 the biggest difficulty in construction of infectious clone is amplification of whole genome, because the 5' end of virus UTR region contains a segment of GC high-content region, it causes great difficulty in sequencing and amplification of virus whole gene, so far, 13 members of circovirus only have the first member of Chicken Infectious Anemia Virus (CIAV) to complete construction of infectious clone, and realize in vivo and in vitro virus rescue, and other 12 viruses do not construct infectious clone and have no clear pathogenicity research. How to fill up the gap becomes a problem to be solved urgently in the field.

Disclosure of Invention

Aiming at the blank existing in the prior art, the invention firstly utilizes the structural characteristics of a circular genome specifically utilizing GyV3 and self-contained enzyme cutting sites, amplifies two sections of virus complete genomes with different starting points by using high-fidelity enzyme, constructs GyV3 double-copy full-length infectious clone pcDNA3.1-2GyV3 by connecting and transforming the virus complete genomes with eukaryotic expression pcDNA3.1(+) through two times, and realizes virus rescue and pathogenicity research in an SPF chicken body; finally, a simple and quick circovirus reverse genetics operating system is provided, a platform is provided for GyV3 pathogenicity and gene structure and function research, and a foundation is laid for researching a GyV3 genetic engineering vaccine.

In 1991, Noteborn and Claessens et al isolated double-stranded replication form (dsRF) DNA of the virus in CIAV-infected LSCC-1104-X-5 and MDCC-MSB1, respectively, and cloned into eukaryotic expression vectors pIC20H and pGEM-7Zf (+), then transfected into MDCC-MSB1 cells, observed cytopathic effect, and detected infectious virus particles in the supernatant by virus neutralization experiments after passage. Noteborn et al continued to inoculate 1 day old SPF chickens with muscle from supernatant of transfected MDCC-MSB1 cells, replicating the same clinical symptoms and histological lesions as the wild strain. In 2000, Brown et al used overlapping PCR amplification to obtain the whole genome of a CIAV Australian isolate and ligated into vector pGEM-4Z, then transfected the recombinant circular vector and enzyme-digested linearized whole genome into MDCC-MSB1 cells, and observed cytopathic effect by indirect immunofluorescence technique (IFA) and inoculating new cells into the supernatant of transfected cells to prove the success of inductive clone construction. In 2011, the plum blossom is used for amplifying CIAV whole genome by using common PCR, 2 full-length genomes are connected to a pBluescript II SK (+) plasmid vector in a forward direction by twice connection transformation through groping the optimal incomplete enzyme digestion condition, and finally the infectious clone construction is successfully verified by IFA in vitro verification and infectious clone plasmid muscle inoculation of 1-day-old SPF chicken for replicating diseases. In 2017, Kaffashi et al used general PCR amplification to obtain the whole genome of a CIAV Israeli isolate and connected to a vector pTZ57R/T, and then the enzyme-digested linearized whole genome was transfected into MDCC-MSB1 cells, and the replication of SPF chickens of 1 day old was observed for cytopathic effect and cell supernatant to prove that CIAV sensitive clones were successfully constructed. In addition, Porcine Circovirus (PCV) also has a precedent for constructing double-copy infectious clones, but because the PCV has no GC high-content region, the acquisition of PCV whole genome and the connection of double copies are simpler.

Therefore, the small and circular gene structure of the circovirus and the dsRF replication characteristic are beneficial to the construction of infectious clones. The construction of the double-copy infectious clone of the invention utilizes the enzyme cutting sites of the virus, avoids the exploration of incomplete enzyme cutting conditions and improves the construction efficiency.

The specific technical scheme of the invention is as follows:

the inventor firstly provides an GyV3 virus strain named as GyV3 SDAU-2 strain, and the inventor carries out biological preservation on the strain, wherein the biological preservation number is CCTCC NO: V202061;

further, the inventor provides an GyV3 double-copy whole genome infectious cloning plasmid, which consists of eukaryotic expression vectors pcDNA3.1(+) and GyV3 double-copy whole genomes and is named as pcDNA3.1-2GyV3, wherein the nucleotide sequence of the double-copy whole genome infectious cloning is shown as SEQ ID NO. 1;

the invention further provides a construction method of the GyV3 double-copy whole genome infectious clone plasmid, which comprises the following specific steps:

artificially designing a primer from 568 th nucleotide by utilizing the characteristics of a virus circular genome, adding HindIII and Not I enzyme cutting sites and corresponding protective bases at the 5' ends of an upstream primer and a downstream primer respectively, placing a GC high-content region in the middle region of an amplified fragment, amplifying a linear whole genome HindIII-GyV 3-Not I by utilizing high-fidelity enzyme, and connecting the linear whole genome HindIII-GyV 3-Not I to pcDNA3.1(+) to construct GyV3 single-copy recombinant plasmid pcDNA3.1-GyV 3; artificially designing a second pair of primers from a BamH I enzyme cutting site (1465 th nucleotide) of a virus genome, directly adding a protective base at the 5' end of the BamH I enzyme cutting site of the virus genome by an upstream primer, and artificially adding a BamH I enzyme cutting site and a protective base by a downstream primer to amplify linear whole genome BamH I-GyV 3-BamH I, connecting the linear whole genome BamH I-GyV 3-BamH I to the obtained pcDNA3.1-GyV3 through second connection and transformation, and finally synthesizing GyV3 double-copy full-length gene infectious clone pcDNA3.1-2GyV 3; according to the scheme, the positions of the enzyme cutting sites and the primers are reselected according to the characteristic of GyV3, and the primer sequences are redesigned, so that the method is obviously different from the conventional technology;

in addition, the inventor also provides the application of the GyV3 double-copy whole genome infectious clone plasmid, and particularly provides a method for verifying infectious clones in vitro, which comprises the following steps: culturing MDCC-MSB1 cells and preparing primary chick embryo renal tubular epithelial cells; transfection of infectious clones; detecting the transcriptional expression condition of the infectious clone by indirect immunofluorescence; more specifically, the constructed GyV3 double-copy full-length gene infectious clone is used for carrying out in-vivo virus rescue on 6 embryo-age yolk sacs, 1 day-age muscles and abdominal cavity inoculated SPF (specific pathogen free) chickens, 7, 14, 21 and 28dpi detection on virus hemorramia and tissue virus load of inoculated chickens is used for verifying whether infectious virus rescue is successful, and then a GyV3 pure infection model is established. Meanwhile, GyV3 tissue isolation virus (GyV3 SDAU-2 strain with the preservation number of CCTCC NO: V202061) is inoculated in the same way as a positive control and PBS is used as a negative control.

The obtained plasmid can be used for GyV3 in vivo virus rescue and pathogenicity research, and related plasmid vectors and other raw materials are cheap and easy to obtain, so that the construction cost and time of infectious clone are greatly shortened. Meanwhile, the technical scheme of the application fills the blank in the field, and the construction strategy of the invention can be used for construction of infectious clone and virus rescue of other circovirus.

Preservation information

Preservation time: 10 and 1 month in 2020

The name of the depository: china center for type culture Collection

The preservation number is: CCTCC NO: V202061

The address of the depository: wuhan university of Wuhan, China

And (3) classification and naming: circovirus type 3 SDAU-2 strain (Gyrovir 3 SDAU-2)

Drawings

FIG. 1 is a schematic diagram of construction of GyV3 double-copy infectious clones;

in the figure, A: GyV3 SDAU-1 genome structure schematic diagram; b: GyV3 construction of a schematic diagram of a double copy infectious clone;

FIG. 2 is a schematic representation of the results of GyV3 whole genome PCR amplification;

in the figure, Phanta Max Master Mix and Vazyme LAmp Master Mix are two high fidelity enzymes; F1/R1 is a first pair of full-length gene amplification primers, and F2/R2 is a second pair of full-length gene amplification primers; yin: normal SPF chicken blood DNA;

FIG. 3 is a diagram showing the screening results of pcDNA3.1-GyV3 positive clones;

in the figure, M is a DNA Marker; 1-10, colony numbering; GyV3 positive chicken blood DNA; negative is pcDNA3.1 empty carrier bacterial liquid;

FIG. 4 is a schematic diagram showing the results of double restriction enzyme identification of pcDNA3.1-GyV3 plasmid;

in the figure, M is a DNA Marker; 2/5/9, numbering the positive cloning plasmid;

FIG. 5 is a diagram showing the screening results of pcDNA3.1-2GyV3 positive clones;

in the figure, M is a DNA Marker; 1-8, numbering bacterial colonies;

FIG. 6 is a schematic diagram showing the single-enzyme digestion identification result of pcDNA3.1-2GyV3 plasmid;

in the figure, M is a DNA Marker; 5/8 plasmid number of positive clone.

FIG. 7 is a gray-scale schematic of the pathological observation and IFA detection results of the MDCC-MSB 1/chicken primary tubular epithelial cell transfected pcDNA3.1-2GyV3 cell;

FIG. 8 is a GyV3 absolute fluorescence quantitative PCR standard curve;

in the figure, a. dissolution curve; B. an amplification curve; C. a standard curve;

FIG. 9 is a graph of the test chicken blood viremia test;

NC in the figure: a normal group; GyV 3-IC: GyV3 infectious clone groups; GyV 3: GyV3 positive control group;

FIG. 10 is a bar chart of the results of virus load detection of organs and feces of experimental chickens.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are intended to illustrate the invention and not to limit it. In this embodiment, except for the special description, the others are all completed by using the prior art.

Example 1 PCR amplification of 1 GyV3 Whole genome

1. Primer design and Synthesis

According to the construction strategy (figure 1), according to GyV3 SDAU-1 nucleic acid sequence of NCBI GenBank database, using SnapGene Viewer software to inquire GyV3 self-contained enzyme cutting sites, two pairs of GyV3 full-length gene amplification primers containing enzyme cutting sites are artificially designed. The underlined part is the cleavage site and the 5' end is the protecting base.

GyV3 full gene amplification primer

Preparation of template DNA of GyV3

GyV3 template DNA was prepared using GyV3-SDAU-2 strain 2 nd-regressive SPF chicken tissues.

GyV3 infected chicken kidney DNA was extracted using a cell/tissue/blood DNA extraction kit (Tiangen, Beijing);

3, GyV3 PCR amplification and purification recovery of the Whole genome

In order to accurately amplify Gy V3 whole genome, PCR was performed using GyV3 DNA samples obtained in the above steps as templates, using 2 XPhanta Max Master Mix and 2 XVazyme LAmp Master Mix (Vazyme, Nanjing) as two high fidelity enzymes. The loading system and PCR reaction procedure are shown in the following table:

PCR sample adding system

PCR reaction procedure

The reaction result was separated and purified by agarose gel electrophoresis (FIG. 2) and the gel was recovered (Tiangen, Beijing) for further use. The results show that Phanta Max Master Mix successfully amplified two whole genomes of the virus: hind III-GyV 3-Not I (SEQ ID NO.6) and BamH I-GyV 3-BamH I (SEQ ID NO. 7).

EXAMPLE 2 construction and characterization of Single-copy full-Length Gene recombinant plasmid pcDNA3.1-GyV3

Double cleavage and ligation of pcDNA3.1(+) and Hind III-GyV 3-Not I

Empty vectors pcDNA3.1(+) and HindIII-GyV 3-Not I were subjected to double digestion using QuickCut HindIII and QuickCut Not I (Takara), respectively, and the following double digestion system was loaded (the amount of empty vectors and HindIII-GyV 3-Not I was Not more than 1. mu.g), reacted in a metal bath at 37 ℃ for 10min, and then the digestion products were purified by agarose gel electrophoresis and recovered for use. The ligation of pcDNA3.1(+) and Hind III-GyV 3-Not I was performed on the same day as the double digestion, and the ligation system was loaded in the following table and reacted at 16 ℃ for 30 min.

Double enzyme digestion system

Connection system

Transformation of pcDNA3.1(+) and HindIII-GyV 3-Not I ligation products

Taking out competent cell DH5 alpha (100 mu L) from a refrigerator at-80 ℃ and directly placing the cell in an ice bath;

adding 10 mu L of a connecting product into the competent cell suspension in a super clean bench (note that the volume of the connecting product does not exceed one tenth of the volume of the competent cell suspension), gently flicking and uniformly mixing, and standing in an ice bath for 30 min;

thirdly, placing the centrifugal tube in a water bath at 42 ℃ for 90s, then quickly transferring the tube to an ice bath to cool the cells for 2.5min, wherein the movement of the centrifugal tube is avoided in the process and is not required to be carried out on a table top with a centrifuge and vortex oscillation work;

adding 900 mul of sterile LB liquid culture medium (without ampicillin) into the centrifuge tube, mixing uniformly, placing the mixture into a shaking table at the temperature of 37 ℃ and the speed of 150rpm, and carrying out shaking culture for 45min to express the ampicillin resistance marker gene on pcDNA3.1(+) plasmid and recover the thallus;

fifthly, mixing the contents of the centrifuge tube uniformly in a super clean bench, sucking 100 mu L of transformed competent cells and dripping the transformed competent cells on an LB solid agar culture medium plate containing ampicillin, burning a triangular glass rod by an alcohol lamp flame for sterilization, slightly and uniformly spreading the cells by the triangular rod until the liquid permeates, and repeating the operation until 10 plates are fully spread. And (3) inverting all the plates, culturing in a constant-temperature constant-humidity incubator at 37 ℃ for 12-16h, and observing the colony state at any time.

Screening and enzyme digestion identification of pcDNA3.1-GyV3 positive clone

Selecting a milky, isolated and large-growing colony from each plate with a small white gun head, placing the colony in 10mL LB liquid culture medium containing ampicillin, placing the test tube in a 37 ℃ shaking table at 220rpm, and performing shaking culture for 12-16h until bacterial liquid OD₆₀₀The culture is stopped when the temperature reaches 2.0-3.0. The screening of pcDNA3.1-GyV3 positive clones was performed directly using the inoculum solution as template and using the loading system and PCR protocol described in example 1. The results showed that of the 10 singles picked, there were 3 whole genes containing GyV3 (FIG. 3).

Plasmid miniextraction kit (Tiangen, Beijing) is used for extracting bacteria liquid plasmid with positive PCR identification, and the specific steps are shown in product specifications.

The extracted plasmid pcDNA3.1-GyV3 is taken for double enzyme digestion identification. The sample was loaded in the double restriction system of example 2 (the amount of empty vector and Hind III-GyV 3-Not I was Not more than 1. mu.g), reacted at 37 ℃ for 10min in a metal bath, and then identified by agarose gel electrophoresis. Agarose gel electrophoresis results showed two fragments of the desired size: the pcDNA3.1 vector and GyV3 full-length gene (deprotected bases) demonstrated successful construction of pcDNA3.1-GyV3 (FIG. 4).

EXAMPLE 3 construction of double-copy full-Length Gene recombinant plasmid pcDNA3.1-2GyV3

pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI digestion and ligation

GyV3 single copy clone plasmids pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI were digested separately with Quickcut BamHI, loaded with the digestion system shown in the following table (the amount of pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI is not more than 1 μ g), reacted in a metal bath at 37 ℃ for 10min, and then purified by agarose gel electrophoresis and recovered with gel for use. The connection of pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI was carried out on the same day as the enzyme digestion, the sample was added according to the following connection system, and the reaction was carried out at 16 ℃ for 30 min. The ligation product was stored at-20 ℃ for further use.

Enzyme digestion system

Connection system

Transformation of pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI ligation products

The transformation of pcDNA3.1-GyV3 and BamHI-GyV 3-BamHI ligation products was performed as in step 2 of example 2

Screening and enzyme digestion identification of pcDNA3.1-2GyV3 positive clone

PCR screening of the bacterial suspension of the positive clone pcDNA3.1-2GyV3 was performed in the same manner as in step 3 of example, and PCR reaction was performed using pcDNA3.1(+) universal primer (upstream) T7-TAATACGACTCACTATAGGG/(downstream) BGH-TAGAAGGCACAGTCGAGG (shown in SEQ ID NO. 8/9) according to the system and procedure shown in the following table, and the results were identified by agarose gel electrophoresis. The results showed that of the 10 singletons picked (of which 2 colonies were not successfully shake-amplified), 2 contained double copies of GyV3 full gene (FIG. 5);

bacteria liquid PCR sample adding system

Bacterial liquid PCR reaction program

Transferring the positive bacteria liquid of the bacteria liquid PCR identification into 200mL of fresh LB liquid culture medium containing ampicillin, placing an erlenmeyer flask in a shaking table at 220rpm and culturing for 12-16h under the condition of 37 ℃ until the OD600 of the bacteria liquid reaches 2.0-3.0, stopping culturing, and extracting bacteria liquid plasmids by using an endotoxin-free plasmid macroextraction kit (Tiangen, Beijing), wherein the specific steps are shown in the product specification.

The extracted plasmid pcDNA3.1-2GyV3 is taken for enzyme digestion identification. The samples were loaded according to the enzyme digestion system in Table 8, reacted in a metal bath at 37 ℃ for 10min, and then identified by agarose gel electrophoresis. The agarose gel electrophoresis results showed two fragments of the expected target size, which confirmed the successful construction of pcDNA3.1-2GyV3 (FIG. 6).

Example 4 validation of in vitro transfection of double copies of the full-Length Gene infectious clone pcDNA3.1-2GyV3

1. Preparation and culture of primary chicken renal tubular epithelial cells

The research of the inventor shows that GyV3 has strong specific signal localization in chicken kidney tubular epithelial cells, so the inventor uses the prior art to make chicken primary tubular epithelial cells, and carries out in vitro transfection verification on pcDNA3.1-2GyV3, and the making and culture processes are as follows:

(1) aseptically taking 18 embryo-aged chicks in a super clean bench, putting the chicks into a disposable aseptic plate, picking the kidneys of the chicks by using aseptic ophthalmic scissors and tweezers, removing connective tissue membranes such as renal vessels and nerves in D' Hanks liquid containing 1% double antibodies as much as possible, and shearing the connective tissue membranes into pieces;

(2) washing with D' Hanks solution for 2 times;

(3) gently wash 1 time with 1% collagenase type I (in serum-free Opti-MEM medium); transferring into a sterilized 50mL centrifuge tube;

(4) adding 1% collagenase I with the volume of about 5 times of the tissue, incubating at 37 deg.C for 40min, and gently shaking the conical flask during incubation;

(5) after the incubation is finished, gently blowing and beating the cells for 30 times by using a 5mL pipette gun, and absorbing and removing the collagenase;

(6) adding a certain amount of D' Hanks liquid, and continuously blowing and beating for 30 times;

(7) filtering with a disposable cell filter with a pore size of 40 μm while fully rinsing the filter with D' Hanks solution and collecting the filtrate;

(8) suspending the filtrate in D' Hanks solution, centrifuging at 800rpm for 8min, and washing for 2 times;

(9) discarding the supernatant, adding 50% percoll cell separation solution, blowing, stirring, centrifuging at 12000rpm at 4 deg.C for 30 min;

(10) sucking the bottommost high-density strip, suspending the high-density strip in D' Hanks liquid (red erythrocyte sediment can be seen at the moment, and taking care not to suck the erythrocytes), blowing and uniformly mixing, centrifuging at 800rpm for 10min, and repeatedly washing for 3 times;

(11) washing with serum-free DMEM for 1 time, and centrifuging at 800rpm for 10 min;

(12) the collected cells were suspended in a chicken epithelial cell complete medium, and the cells were counted at 2.5X 10⁶One cell/well was inoculated into a 6-well cell culture plate, another T25 cell flask was inoculated for passaging, placed at 37 ℃ with 5% CO₂Culturing in a cell culture box for later use.

Subculture of MDCC-MSB1 cells

Whereas most CIAV strains can be stably propagated in MDCC-MSB1, MDCC-MSB1 (a commercial cell line, purchased from BNCC (beijing)) used by the inventors was subjected to in vitro transfection validation of pcdna3.1-2GyV3, with cell passage and plating steps as follows:

(1) when the original cell culture solution turns yellow and becomes light, or the cells are observed under a microscope to have an overlapping phenomenon, passage can be carried out. Incubating PBS, 1640 culture medium and fetal calf serum in a 37 deg.C water bath for 30 min;

(2) transferring the cell suspension liquid in the T25 cell bottle into a sterilized 10mL centrifugal tube in a super clean bench, and centrifuging for 10min at 800 rpm;

(3) adding 3mL of PBS, gently blowing and beating the suspension cells, centrifuging at 800rpm for 10min, and repeatedly washing for 2 times;

(4) adding a proper amount of 4mL 1640 culture medium containing 10% fetal calf serum, gently blowing and beating to obtain suspension, uniformly packaging into 2T 25 cell bottles with 2mL each, and supplementing the culture medium to 5mL each bottle; place the cell vial in a 5% CO solution₂Culturing in a cell culture box at 37 ℃;

(5) at a proper time, well-activated cells in logarithmic growth phase are plated in a six-well plate, 2mL of 1640 medium containing 10% fetal bovine serum is added to each well, and the cells are cultured in a cell culture box for later use.

3. Transfection of the infectious clone pcDNA3.1-2GyV3

(1) When the confluence degree of the primary chicken renal tubule cells and MDCC-MSB1 cells respectively reaches about 70 percent, performing transfection;

(2) the Roche Lipofectin HP DNA Transfection Reagent, the plasmid (empty vector pcDNA3.1(+), infectious clone plasmid pcDNA3.1-2GyV3) and Opti-MEM were removed from the refrigerator and placed at room temperature, equilibrated to +15 to +25 ℃. Mixing the X-tremeGENE HP DNA Transfection Reagent by short vortex;

(3) taking 3 sterile 1.5mL centrifuge tubes, diluting plasmids in two centrifuge tubes by using Opti-DMEM (Opti-modified eagle-tube to make the final concentration of the final-modified eagle-tube to be modified eagle-segment-modified eagle-segment of each of 3 sterile 1.each of each of the empty; adding 400 mu L of Opti-DMEM into the 3 rd centrifugal tube;

(4) adding 3 μ L of X-tremagene HP DNA Transfection Reagent directly into 3 centrifuge tubes, directly injecting into the liquid below the liquid level, and gently mixing without contacting the Transfection Reagent with the plastic tube wall;

(5) incubating the transfection reagent, DNA complex, at room temperature for 15 min;

(6) taking out the 6-hole cell culture plate from the cell culture box, directly and dropwise adding the transfection compound into cells without changing a culture medium, wherein each hole is 100 mu L;

(7) the plate is gently shaken or rotated by hand to distribute the transfection complex evenly over the surface of the plate, and then returned to the cell incubator for further incubation for 72 h.

4. Indirect immunofluorescence detection of protein expression

Taking the 6-well plate cell incubated in the previous step, and performing indirect immunofluorescence detection on the transcriptional expression of pcDNA3.1-2GyV3 in vitro cells by using rabbit source anti-GyV 3 VP1 multi-antiserum (GyV3 is referred to Yuan. circovirus type 3. research on the pathogenic characteristics of chicken and mice [ D ]. Shandong agricultural university, 2020. published method). Due to the difference in growth characteristics of the two cells, the previous cell fixation procedure is different:

MDCC-MSB1 belongs to complete suspension cells, when in fixation, cell suspension of each hole is taken in a 2mL sterilization centrifugal tube, centrifugation is carried out for 10min at 1000 r/min for precipitating cells, supernatant is collected aseptically, the precipitated cells are washed twice by PBS, a small amount of PBS is used for suspending the cells, then the cells are respectively coated in the center of a new 6-hole plate, and after the cells are dried, acetone-ethanol 3: 2 mixed fixation solution precooled at minus 20 ℃ is used for fixation for 10min at room temperature.

The primary chicken renal tubular epithelial cells belong to adherent cells, after supernatant is collected aseptically, the adherent cells are washed twice by PBS, 2mL of PBS is added into each hole for washing for 5 minutes on a shaking table, and then the same acetone-ethanol precooling fixing solution is added for fixing for 10 minutes. The antibody incubation operation for the two cells was then the same: removing the fixative from each well, washing the cells in each well with PBS for 3 times, each time for 3 min; adding 500 mu L of rabbit source anti-GyV 3 VP1 multi-antiserum diluted 1: 100 into each well, and incubating for 1 hour at 37 ℃; washing with PBS for 3 times, each for 3 minutes; adding 500 mu L of FITC-labeled goat anti-rabbit IgG diluted by 1: 300 into each hole, wrapping a transparent cell plate by using tinfoil paper, keeping out of the sun, and incubating in a constant-temperature constant-humidity incubator at 37 ℃ for 1 hour; washing with PBS in dark for 3 times, each for 3 minutes; the fluorescence was photographed by observation with an inverted fluorescence microscope.

The results showed that the expression of pcDNA3.1-2GyV3 VP1 could be detected in both transfected cells and that cell swelling, ruptured cytopathic effect could be observed (FIG. 7).

EXAMPLE 5 construction of 5 GyV3 Absolute fluorescence quantitation Standard Curve

In order to detect the viremia level and the tissue virus load of the chicken in the infection model of the infectious clone pcDNA3.1-2GyV3 to verify whether the virus is successfully rescued, an absolute fluorescent quantitative detection method aiming at GyV3 gene needs to be constructed.

An absolute fluorescent quantitative PCR Primer was designed using GyV3 SDAU-1 strain VP1 gene as a template and Primer 6.0, and the Primer sequences are shown in the following table.

GyV3 Absolute fluorescence quantitative primer sequence

Taking the plasmid pcDNA3.1-GyV3 constructed and extracted in the example 2 as a standard substance, measuring the concentration of the standard substance, and converting the concentration of the plasmid into the copy number according to the formula:

plasmid copy number (copies/. mu.L) ═ Avgalois constant 6.02X 10²³(copies/mol). times.plasmid concentration (ng/. mu.L). times.10^-9/[ (vector molecular weight + insert molecular weight). times.660](g/mol), calculating the copy number of the standard product.

The plasmid standard was diluted 1X 10 by double dilution with double distilled water⁹-1×10³3 parallel repeats are set for each gradient, 3 technical repeats are set for each repeat, a fluorescent quantitative PCR reaction system and a two-step amplification program are respectively shown in tables 12 and 13, the reaction is carried out on a Roche fluorescent quantitative PCR instrument, and the instrument is provided with analysis software to automatically generate a standard curve. As a result, GyV3 absolute fluorescence quantitative standard curve was successfully constructed.

FIG. 8A shows a single peak in the melting curve, indicating that the qPCR primers used in the present invention have good sensitivity and specificity and can be used for the GyV3 standard curve establishment; FIG. 8B shows that the amplification curves have distinct peaks and known intervals, which indicates that the standard substance has accurate dilution and good repeatability; the standard curve equation for this absolute fluorescence quantification is obtained from FIG. 8C as: Y-2.7465X +34.54 (Y: Ct value, X: template initial copy number), regression coefficient R2-0.9951, indicating that the curve has good correlation and can be used for absolute quantitative analysis of GyV 3.

Example 6 GyV3 in vivo Virus rescue and establishment of model of SPF Chicken infection

The establishment of infectious clone pcDNA3.1-2GyV3 infection model (strain GyV3 SDAU-2) is carried out by adopting 6 embryo age yolk sac inoculation, 1 day age abdominal cavity inoculation and muscle inoculation to ensure the full action of infectious clone plasmid and in vivo cells. And (3) randomly selecting 3 chickens per group for blood sampling and cloaca swab killing 7, 14, 21 and 28 days after the muscle abdominal cavity inoculation, and carrying out virus rescue detection. Grouping and inoculation protocols are summarized in the following table

GyV3 grouping and inoculation mode for virus rescue experiment in infectious clone body

Viremia test is an important index for testing whether virus infectious clone has infectivity, and the invention monitors the blood virus load level of all chickens in 7dpi each group and random 3 chickens in 14/21/28dpi each group. As a result, it was found that both GyV3 infectious clone group and positive control group had persistent viremia during the experimental period, in which the GyV3-IC group blood viral load peaked at 14dpi and then declined, and the GyV3 group blood viral load rose continuously to 21dpi and then also showed a declined trend (FIG. 9). This indicates that the double-copy full-length gene infectious clone pcDNA3.1-2GyV3 achieves in vivo viral rescue and produces effective infection of SPF chickens.

Using the absolute fluorescence quantitative standard curve successfully constructed in example 5, 12 tissue sample DNAs of each group of heart, liver, spleen, lung, kidney, brain, glandular stomach, duodenum, bone marrow, thymus, bursa of fabricius and cloacal swab (feces) were extracted, and quantitative PCR detection was performed at four time points of 7dpi, 14dpi, 21dpi, and 28 dpi.

The results show (fig. 10): the dynamic trend of viral load was substantially consistent for each tissue of the four time points GyV3-IC and GyV3 groups. Wherein the 7dpi bone marrow has the highest virus load, and then has the kidney, bursa of fabricius, thymus and spleen, and the heart, liver, lung, glandular stomach, duodenum and feces have lower loads; the 14dpi kidney has the highest virus load, the glandular stomach and the liver have the lowest virus load, and the feces virus load is higher than 7 dpi; the renal viral loads of 21dpi and 28dpi remain highest, followed by bursa of fabricius, thymus and bone marrow, and other tissues and stool viral loads remain low. From the overall viral load of the four time points, the viral load is highest at 7 and 14dpi, the viral load can reach 7.92 multiplied by 108copies/g, and the viral load of 21 and 28dpi is in overall descending trend; from the viral load of cloacal swabs, both GyV3-IC and GyV3 groups were able to excrete virus through the cloacal cavity, with the highest viral load at 14dpi, and also directly indicate that the infectious clones of the invention can synthesize intact virions in vivo.

Sequence listing

<110> Shandong university of agriculture

<120> circovirus type 3 double-copy full-length gene infectious clone plasmid, construction method and application thereof

<150> 2021104703754

<151> 2021-04-29

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4712

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ccaggtcggt gatattcgag catccaataa gttcgtcgga gtcggttggg actctctcca 60

aagagatcca aattgggctc gggtcaacta taattaccgt atcgcttcct ggcttcgcga 120

gtgttcgcgt actcacgacg cgatctgcaa ctgcgggggc ttcagacgcc actggttcca 180

ggaggcagca ggactgtcca cacaggagac ccagacggac ccggtcgcca gagatctcga 240

tcgcctggtc gtgcgtggaa acgcagcaaa aagaaaattg gattacatcg cgaacagaaa 300

aactcccaaa aagagaaagg ctaagactgt aacatggctc gacgatttcg ccggcacaga 360

ggaaagttcg gatactacag acggggaaga tggcactgga gacacagact gcgacgaaga 420

cgctattccc ggaggcgtaa acttcgatat gcgcgtcgac gacccagtgc tcgcagcgtt 480

aaaaggaaga tattcaaccc acatccggga tcttacctgg taagactgcc gaacccctat 540

aacgcgatta acctatactt tcaagggctc gtattcatac ctagagccac aagctactta 600

ccagacacaa ctaaaggcaa aaacgttaca acaactaatg tggcactaat taacgttaac 660

ctgaaagagt tcttctgggc cacactgcca ctagacgcaa ggtcaaagat tggaggaccc 720

aaccccttcc cacaacacat ccagggatgt gactgggcgg gcatagccac aacccacaaa 780

ggctgctggc catacagtac acaaatgtca tcatctagac agccaggggc atggccttca 840

gaatggtggc gatgggcact tcttcttatg catcctagat ccaatgtacg attcttcgga 900

tccccgaaac tgatgaccct accacaaata ggacagttcc tggggggctg gcaactattc 960

acccacagat tcacaaaatt ccgtgtgctt gcaactaaga gcagagaatc gttctccccg 1020

gtcgcgagcc tgcttgtaca agacaattac tttgcaagaa gagagggtgc agggccacca 1080

atatcgggac aaccaccaat gtgcaccatg caaagactta cgagagacta tacaggcaca 1140

gaaagcaatg ctccagctaa tgaaaccaca ataccatcca tgccaccaga cccaccccaa 1200

taccccgctc aaaccggctg cagcacggcg gtagaccctg gtgaatacct cctcgcagga 1260

ctcacacgta cagcagtatc ctgctggtat tcacgctcaa catacccaag ctttgctacg 1320

ctatcagcac taggggcacc atggtcattc ccagcaggac agaagtcaat cagcaaaaca 1380

tccttcaaca aacatgtcat tagaggcatg ggtgacccac aaggcaaaaa atggctcacc 1440

ctggtaccga aagaacaaga atggatcaat tcggactcaa tgacaaagtc agaactggac 1500

acggacatag ctacattgta cctagctcaa ggaacaagca gagcaaacag ctacaaattc 1560

aacacattcc acgaggtaat ggtacaagac cccatgaatg tagccccctg ggcagtcgtc 1620

aaagtctcca gcgtctggac actcggcaac aacagaagac catacccatg ggatgtcaac 1680

tggtacaacg aattcactgc agaaggccgc gtccccgcag actagggggg ggggggaaaa 1740

cccccccctg accccccccc cgggggggat cttccccccc gaacccccca tctgtcaata 1800

aaaagctttt tacaaactac aagtgtttac tattctgtca cccttctagc atgtacacaa 1860

aaagtcaaga tggacaaatc gctcgacttc gctcgcgatt tgtcgaaggc ggggggccgg 1920

aggccccccg gtggcccccc gccaactggg gaagcgtgta cacaaagata acgcgaaccg 1980

cacccactag tgacgtaccc ccctgtacag cgtgacgtac ccccctgtac atgggcggga 2040

ctgtacaggg gggtacgtca tccttatctc caaccaatag agccgctccc cgcccacagg 2100

gcgggcagat catatcgcgc aaagcataaa aagcgcgtac taaccggacg gcagaaggta 2160

tgtcatccgg cggtctaggt gatagttcgg agaacgagca actagccgct gggggcagtg 2220

aattgccgct taggcaagag gggcaacttg ggcccagcgg agccggatca acgggcaaga 2280

aacttaaaaa gcacgactct ccctacctga atggaaccgg gacttggaca ccagacccca 2340

agaactacag aaccatccag gtcggtgata ttcgagcatc caataagttc gtcggagtcg 2400

gttgggactc tctccaaaga gatccaaatt gggctcgggt caactataat taccgtatcg 2460

cttcctggct tcgcgagtgt tcgcgtactc acgacgcgat ctgcaactgc gggggcttca 2520

gacgccactg gttccaggag gcagcaggac tgtccacaca ggagacccag acggacccgg 2580

tcgccagaga tctcgatcgc ctggtcgtgc gtggaaacgc agcaaaaaga aaattggatt 2640

acatcgcgaa cagaaaaact cccaaaaaga gaaaggctaa gactgtaaca tggctcgacg 2700

atttcgccgg cacagaggaa agttcggata ctacagacgg ggaagatggc actggagaca 2760

cagactgcga cgaagacgct attcccggag gcgtaaactt cgatatgcgc gtcgacgacc 2820

cagtgctcgc agcgttaaaa ggaagatatt caacccacat ccgggatctt acctggtaag 2880

actgccgaac ccctataacg cgattaacct atactttcaa gggctcgtat tcatacctag 2940

agccacaagc tacttaccag acacaactaa aggcaaaaac gttacaacaa ctaatgtggc 3000

actaattaac gttaacctga aagagttctt ctgggccaca ctgccactag acgcaaggtc 3060

aaagattgga ggacccaacc ccttcccaca acacatccag ggatgtgact gggcgggcat 3120

agccacaacc cacaaaggct gctggccata cagtacacaa atgtcatcat ctagacagcc 3180

aggggcatgg ccttcagaat ggtggcgatg ggcacttctt cttatgcatc ctagatccaa 3240

tgtacgattc ttcggatccc cgaaactgat gaccctacca caaataggac agttcctggg 3300

gggctggcaa ctattcaccc acagattcac aaaattccgt gtgcttgcaa ctaagagcag 3360

agaatcgttc tccccggtcg cgagcctgct tgtacaagac aattactttg caagaagaga 3420

gggtgcaggg ccaccaatat cgggacaacc accaatgtgc accatgcaaa gacttacgag 3480

agactataca ggcacagaaa gcaatgctcc agctaatgaa accacaatac catccatgcc 3540

accagaccca ccccaatacc ccgctcaaac cggctgcagc acggcggtag accctggtga 3600

atacctcctc gcaggactca cacgtacagc agtatcctgc tggtattcac gctcaacata 3660

cccaagcttt gctacgctat cagcactagg ggcaccatgg tcattcccag caggacagaa 3720

gtcaatcagc aaaacatcct tcaacaaaca tgtcattaga ggcatgggtg acccacaagg 3780

caaaaaatgg ctcaccctgg taccgaaaga acaagaatgg atcaattcgg actcaatgac 3840

aaagtcagaa ctggacacgg acatagctac attgtaccta gctcaaggaa caagcagagc 3900

aaacagctac aaattcaaca cattccacga ggtaatggta caagacccca tgaatgtagc 3960

cccctgggca gtcgtcaaag tctccagcgt ctggacactc ggcaacaaca gaagaccata 4020

cccatgggat gtcaactggt acaacgaatt cactgcagaa ggccgcgtcc ccgcagacta 4080

gggggggggg ggaaaacccc cccctgaccc cccccccggg ggggatcttc ccccccgaac 4140

cccccatctg tcaataaaaa gctttttaca aactacaagt gtttactatt ctgtcaccct 4200

tctagcatgt acacaaaaag tcaagatgga caaatcgctc gacttcgctc gcgatttgtc 4260

gaaggcgggg ggccggaggc cccccggtgg ccccccgcca actggggaag cgtgtacaca 4320

aagataacgc gaaccgcacc cactagtgac gtacccccct gtacagcgtg acgtaccccc 4380

ctgtacatgg gcgggactgt acaggggggt acgtcatcct tatctccaac caatagagcc 4440

gctccccgcc cacagggcgg gcagatcata tcgcgcaaag cataaaaagc gcgtactaac 4500

cggacggcag aaggtatgtc atccggcggt ctaggtgata gttcggagaa cgagcaacta 4560

gccgctgggg gcagtgaatt gccgcttagg caagaggggc aacttgggcc cagcggagcc 4620

ggatcaacgg gcaagaaact taaaaagcac gactctccct acctgaatgg aaccgggact 4680

tggacaccag accccaagaa ctacagaacc at 4712

<210> 2

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

ccaagcttcc aggtcggtga tattcg 26

<210> 3

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atttgcggcc gcatggttct gtagttcttg g 31

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gggatccccg aaactgatg 19

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cgggatccga agaatcgtac attgg 25

<210> 6

<211> 2356

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6