阅读说明：本技术 一种表达非洲猪瘟病毒CD2v蛋白的细胞株及其应用 (Cell strain for expressing African swine fever virus CD2v protein and application thereof ) 是由 李红卫 颜仁和 王升启 毛莹莹 *** 高永新 于 2019-10-30 设计创作，主要内容包括：本发明属于生物医药基因工程与免疫学领域,具体涉及一种表达非洲猪瘟病毒CD2v蛋白的细胞株及其应用。本发明提供的表达非洲猪瘟病毒CD2v蛋白的细胞株,是通过对非洲猪瘟CD2v蛋白核酸序列进行优化合成,构建含ASFV-CD2v基因的重组慢病毒载体,然后包装慢病毒并感染HEK-293T细胞,再经筛选获得的。本发明提供的表达非洲猪瘟病毒CD2v蛋白的细胞株,具有与亲本细胞相似的生物特性,有利于抗原蛋白的规模生产；表达蛋白在表达细胞内能得到接近病毒蛋白的天然构象与修饰加工,抗原性好,易于大量生产。(The invention belongs to the fields of biomedical genetic engineering and immunology, and particularly relates to a cell strain for expressing African swine fever virus CD2v protein and application thereof. The cell strain for expressing the African swine fever virus CD2v protein is obtained by optimizing and synthesizing an African swine fever CD2v protein nucleic acid sequence, constructing a recombinant lentivirus vector containing an ASFV-CD2v gene, packaging lentivirus, infecting HEK-293T cells and screening. The cell strain for expressing the African swine fever virus CD2v protein has the biological characteristics similar to those of parent cells, and is beneficial to the scale production of antigen protein; the expressed protein can obtain natural conformation and modification processing similar to those of virus protein in the expression cell, has good antigenicity and is easy for mass production.)

1. A recombinant African swine fever virus CD2v protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. The recombinant african swine fever virus CD2v protein of claim 1, wherein the sequence encoding the amino acid is a polynucleotide sequence.

3. The recombinant african swine fever virus CD2v protein according to claim 2, wherein the polynucleotide sequence information encoding the amino acid sequence is as set forth in SEQ ID No. 2.

4. The recombinant african swine fever virus CD2v protein according to claim 3, wherein the upstream primer information for amplifying the polynucleotide sequence of the amino acid sequence is as set forth in SEQ ID No. 3; the downstream primer information of the polynucleotide sequence for amplifying the amino acid sequence is shown as SEQ ID No. 4.

5. A recombinant cell strain capable of efficiently and stably secreting and expressing African swine fever virus CD2v protein is characterized in that the recombinant cell strain is preserved in China center for type culture collection with the preservation strain number of HEK-293T-CD2 v.

6. An application of a recombinant African swine fever virus CD2v protein in diagnosing African swine fever immune serum.

7. An application of a recombinant African swine fever virus CD2v protein in the aspect of immune mucosal adjuvant.

Technical Field

The invention belongs to the fields of biomedical genetic engineering and immunology, and particularly relates to a cell strain for expressing African swine fever virus CD2v protein and application thereof.

Background

African Swine Fever (ASF) is an acute, febrile and highly contagious disease of pigs caused by African Swine Fever Virus (ASFV), the clinical symptoms and pathological changes of the ASFV are similar to those of acute swine fever, high fever, skin congestion, abortion, edema and organ bleeding are shown, and the lethality rate can reach 100%. The disease is mainly epidemic in Africa at first, gradually spreads to countries and regions such as Europe and Asia in recent years, after the first sick pig appears in 2018 of China, the death rate of infected pigs is close to 100% and huge loss is caused to the pig raising industry in China, wherein the first sick pig is reported in Liaoning, Henan, Jiangsu, Zhejiang, Anhui, Heilongjiang, inner Mongolia, Jilin and other provinces in several months. China already defines the disease as a type of animal disease, and the world animal health Organization (OIE) classifies the disease as a necessity animal epidemic disease, which is highly valued by countries in the world.

The ASFV virion is an icosahedral structure consisting of virus genome DNA wrapped by nucleocapsid protein, virus inner envelope, virus capsid and outer envelope, and has a diameter of about 200 nm. The envelope protein is the main structural protein constituting the virus particle, is also an important surface antigen, and is closely related to host cell tropism, pathogenicity and immunogenicity. The functional ASFV envelope proteins are mainly known as CD2v, p54, p12, p30, p17, p22 and the like.

Because of the high lethality and infectivity of ASF and the temporary absence of effective vaccines, strict hygiene and biosafety control measures are required to prevent the spread of the disease, which relies on a rapid, reliable early diagnosis of the disease. Currently, laboratory diagnostics of ASF include methods of animal vaccination, virus isolation, viral nucleic acid DNA detection, and specific antibody detection. However, detection methods such as animal inoculation, virus isolation, and virus nucleic acid DNA require laboratories with more than three levels of biosafety, specialized instruments and technicians, or are cumbersome to operate, which makes it difficult to meet the needs of basic quarantine departments. Therefore, it is necessary to establish a rapid and safe diagnosis and quarantine method using the recombinant ASFV antigen. In recent years, with the completion of sequencing of the whole genome of ASFV, researchers at home and abroad express part of important structural proteins (such as P30, P54, P72, CD2v and the like) of ASFV by using genetic engineering technology, and use the expressed recombinant protein for research on antigens for serodiagnosis of ASFV.

Chinese patent application CN110269932A discloses an African swine fever vaccine and application thereof, and specifically discloses an immunogenic composition, which comprises a first construct, a second construct and a third construct, the composition can be used for preparing an ASFV vaccine or a medicament for treating or preventing ASFV infection, the vaccine reduces the risk of infecting African swine fever virus in the pig raising process to a certain extent, and solves the problem that no African swine fever vaccine exists in China. However, the vaccine preparation process is extremely complicated and the corresponding antigen production is low, which ultimately results in low suppression of african swine fever.

In conclusion, the problems of low antigen yield, complex detection method, poor inhibition on African swine fever and the like generally exist in the prior art.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a cell strain for expressing African swine fever virus CD2v protein and application thereof. The cell strain for expressing the African swine fever virus CD2v protein is stable in expression, still keeps good protein expression level after multiple passages, and has high expression quantity of corresponding protein antigens.

In order to achieve the purpose, the invention adopts the technical scheme that:

a recombinant African swine fever virus CD2v protein has an amino acid sequence shown in SEQ ID NO. 1.

MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNT LATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTI KLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWN NSNINNFTATCIINNTISTSNETTLINCTYLTLSSNYFYTFFKLYHHHHHH(SEQID NO.1)。

Preferably, the sequence encoding the amino acid is a polynucleotide sequence.

More preferably, the polynucleotide sequence information encoding the amino acid sequence is shown in SEQ ID NO. 2.

ATGATAATACTTATTTTTTTAATATTTTCTAACATAGTTTTAAGTATTGATT ATTGGGTTAGTTTTAATAAAACAATAATTTTAGATAGTAATATTACTAATGATA ATAATGATATAAATGGAGTATCATGGAATTTTTTTAATAATTCTTTTAATACAC TAGCTACATGTGGAAAAGCAGGTAACTTTTGTGAATGTTCTAATTATAGTAC ATCAATATATAATATAACAAATAATTGTAGCTTAACTATTTTTCCTCATAATGA TGTATTTGATACAACATATCAAGTAGTATGGAATCAAATAATTAATTATACAAT AAAATTATTAACACCTGCTACTCCCCCAAATATCACATATAATTGTACTAATT TTTTAATAACATGTAAAAAAAATAATGGAACAAACACTAATATATATTTAAAT ATAAATGATACTTTTGTTAAATATACTAATGAAAGTATACTTGAATATAACTG GAATAATAGTAACATTAACAATTTTACAGCTACATGTATAATTAATAATACAA TTAGTACATCTAATGAAACAACACTTATAAATTGTACTTATTTAACATTGTCA TCTAACTATTTTTATACTTTTTTTAAATTATATCACCACCACCACCATCACTG (SEQ ID NO.2)。

Preferably, the upstream primer information of the polynucleotide sequence for amplifying the amino acid sequence is shown as SEQ ID No. 3; the downstream primer information of the polynucleotide sequence for amplifying the amino acid sequence is shown as SEQ ID No. 4.

ATCGATTACTGGGTGTCCTT(SEQ ID NO.3)；

TGTTGATGTTGGAGTTGTTC(SEQ ID NO.4)。

The invention also provides a recombinant cell strain capable of efficiently and stably secreting and expressing African swine fever virus CD2v protein, which is preserved in China center for type culture collection, the address is China, Wuhan university, the name of the culture is mammalian cell HEK-293T, the number of the preserved strain is HEK-293T-CD2v, the name of the Chinese classification is human embryo kidney cell HEK-293T-CD2v, the preservation number is CCTCC NO. C2019275, the preservation date is 2019, 10 and 29 days, and the preservation period is 30 years.

The invention also provides application of the recombinant African swine fever virus CD2v protein in diagnosing African swine fever immune serum, which adopts an indirect ELISA method to detect African swine fever.

The invention also provides application of the recombinant African swine fever virus CD2v protein in immune mucosal adjuvant. The invention unexpectedly discovers that the vaccine prepared by using the CD2 v-expressing protein as an immune mucosal adjuvant in combination with Porcine Epidemic Diarrhea Virus (PEDV) S1 protein and 206 adjuvant can induce an animal body to generate a high titer IgA antibody aiming at the PEDV and resist virus infection; the breakthrough progress plays a key role in the development of the PEDV S1 subunit vaccine, the vaccine is expected to be applied to the clinic to thoroughly control the generation of PED, and the intramuscular injection immunization route is also favorable for the clinical popularization of the vaccine.

The invention is optimized and synthesized on the basis of the known CD2v protein nucleic acid sequence of the African swine fever on GENBANK, constructs a recombinant lentivirus vector containing ASFV-CD2v gene, then packages lentivirus and infects HEK-293T cells, screens to obtain a recombinant cell line capable of stably expressing CD2v protein, produces and purifies to obtain the CD2v protein. The obtained recombinant protein is used as an antigen for detecting the African swine fever, and an African swine fever serum immunological detection method is established; the recombinant porcine epidemic diarrhea virus vaccine is used as an immune mucosal adjuvant and is combined with the porcine epidemic diarrhea virus vaccine (application number: 201610573424.6, a recombinant cell line for stably expressing the porcine epidemic diarrhea virus S1 protein, a vaccine and application), and the high-level IgA antibody can be generated in piglets by intramuscular injection of Tibet miniature pigs.

Meanwhile, the invention constructs a recombinant cell line capable of expressing the African swine fever CD2v protein by using a lentiviral vector, expresses the recombinant protein CD2v by using the cell line, and researches the application of the cell line in immune serum diagnosis and the effect of the cell line as an immune mucosal adjuvant in porcine epidemic diarrhea virus vaccines. The preparation method lays a foundation for the preparation and prevention control of an immune serological diagnostic reagent for African swine fever in China, finds an immune mucosal adjuvant which can generate a high-level IgA antibody by intramuscular injection of a porcine epidemic diarrhea virus vaccine, and plays a key role in the research and development of a PEDV subunit vaccine.

Compared with the prior art, the recombinant cell strain for expressing the African swine fever CD2v protein has the technical advantages that:

(1) the protein expression system selected by the invention is a mammalian cell HEK-293T, and the obtained recombinant cell line HEK-293T-CD2v has biological characteristics similar to those of a parent cell, and is beneficial to large-scale production of antigen protein; the expressed protein can obtain natural conformation and modification processing close to the virus protein in an expression cell, and has good antigenicity; the recombinant cells can be subjected to high-density fermentation culture by using a bioreactor, and are easy for mass production;

(2) according to the invention, a lentivirus vector is adopted to carry a target gene to construct a recombinant plasmid, then HEK-293T cells are transfected, and the generated high-titer virus particles infect the HEK-293T cells again so as to construct an obtained recombinant cell line, which is stable in expression and still maintains a good protein expression level after multiple passages;

(3) according to the invention, a lentivirus expression system is used for expressing the CD2v protein for the first time, and the sequence also contains a histidine tag sequence, so that the later purification is facilitated, and the cost is lower;

(4) the recombinant cell strain provided by the invention does not need African swine fever virus in the process of producing protein, has good biological safety, and the produced protein has good immunogenicity, high sensitivity, strong specificity, high accuracy and extremely high antigen expression quantity, and lays a foundation for accurate diagnosis of African swine fever.

Drawings

FIG. 1 is an enzyme cutting electrophoresis diagram of a lentivirus recombinant vector pLV-CMV-CD2 v;

FIG. 2 is a PCR electrophoresis diagram of a lentiviral recombinant vector pLV-CMV-CD2 v;

FIG. 3 shows the results of the detection of E2 protein expression by different clone cells after transfection;

FIG. 4 shows the results of protein expression detection of different generation subcells E2 after screening clone cell lines;

FIG. 5 shows the RT-PCR results of E2 gene of different generations of recombinant cell line;

FIG. 6 is a protein purification assay;

FIG. 7 is a graph of IgA levels in pig serum, feces, and saliva measured before and after immunization;

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The lentiviral packaging helper plasmids gag/pol, Rev1.66 and VSVG were all purchased from Invitrogen.