Preparation method of porcine deltacoronavirus recombinant N protein and preparation method of polyclonal antibody of porcine deltacoronavirus recombinant N protein

A coronavirus and protein technology is applied in the field of preparation of recombinant N protein of porcine delta-coronavirus and its polyclonal antibodies, which can solve the problems of less research on PDCoV antibody detection methods.

Inactive Publication Date: 2019-04-19
GUANGXI VETERINARY RES INST
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Problems solved by technology

[0004] At present, the diagnostic method for PDCoV is mainly RT-PCR method, and there are few studies on the detection method for PDCoV antibody

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  • Preparation method of porcine deltacoronavirus recombinant N protein and preparation method of polyclonal antibody of porcine deltacoronavirus recombinant N protein
  • Preparation method of porcine deltacoronavirus recombinant N protein and preparation method of polyclonal antibody of porcine deltacoronavirus recombinant N protein
  • Preparation method of porcine deltacoronavirus recombinant N protein and preparation method of polyclonal antibody of porcine deltacoronavirus recombinant N protein

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[0033] 1Materials and methods

[0034] 1.1 Main test materials

[0035] Porcine D-Coronavirus (CH / GX / 1468B / 2017), Porcine D-Coronavirus Negative Serum, and Porcine D-Coronavirus Artificially Infected Pig Positive Serum were separated, prepared and preserved by the applicant's laboratory. Viral genomic DNA / RNA rapid extraction kit is a product of Axygen Biotechnology Co., Ltd.; Taq Mix, DNA gel recovery kit, and plasmid small amount extraction kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; pMD18-T Vector, BamHI, HindⅢ, and DL2000DNAMarker are all products of Dalian Bao Bioengineering Co., Ltd.; pre-stained protein Marker (CW0986), pre-stained protein Marker (CW2841), His tag protein purification kit (CW0894S) are products of Kangwei Century Company; HRP labeled Goat anti-pig IgG antibody is a product of the American company Earthox; E.coli DH5α, E.coli BL21 strains, and the prokaryotic expression vector pET-32a(+) are kept in the applicant's laboratory.

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Abstract

The invention discloses a preparation method of a porcine deltacoronavirus (PDCoV) recombinant N protein. The method comprises the steps that the N gene segment of the PDoV is amplified, subjected toprokaryotic expression and purified in sequence, and the recombinant N protein is obtained. On this basis, the invention further provides a preparation method of a polyclonal antibody of the recombinant N protein. The method comprises the steps that the PDoV recombinant N protein and the Freund's adjuvant are emulsified to immunize animals to obtain polyclonal antibody serum. Through experiments,it is shown that the N gene of the PDCoV is successfully amplified, and the amplified N gene is subjected to prokaryotic expression and purified, the immunogenicity of the recombinant N protein is primarily studied, and a basis is laid for developing a diagnostic kit or preparing the polyclonal antibody.

Description

Technical field [0001] The invention belongs to the technical field of porcine D coronavirus, and in particular relates to a method for preparing a porcine D coronavirus recombinant N protein and its polyclonal antibody. Background technique [0002] Porcine deltacoronavirus (PDCoV) was first discovered in 2012. Woo of the University of Hong Kong and others detected porcine deltacoronavirus in healthy commercial pigs. Coronaviruses are divided into four subgroups: A, B, C, and D. Coronavirus D (PDCoV) that infects pigs is the only D coronavirus that infects non-avian species. Since then, the epidemic of PDCoV has been reported in the United States, China, South Korea and many other countries. Similar to PEDV and TGEV, PDCoV mainly causes intestinal diseases in pigs, leading to diarrhea, vomiting and dehydration in infected pigs. Morbidity and mortality can be as high as 50%-100%, especially in suckling stage piglets. The health of the herd has brought huge economic losses to th...

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Application Information

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IPC IPC(8): C07K14/165C12N15/70C07K16/10
CPCC07K14/005C07K16/10C12N15/70C12N2770/20022C12N2770/20051
Inventor 秦毅斌刘磊卢冰霞李斌周英宁段群棚何颖赵武梁家幸蒋冬福卢敬专
Owner GUANGXI VETERINARY RES INST
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