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Application of SVA 3C protein in promotion of porcine virus replication

A virus replication, pig-derived technology, applied in the direction of double-stranded DNA virus, positive-sense single-stranded RNA virus, application, etc., can solve the problems of reduced immune protection effect of vaccine, weak virulence, limited and other problems, to improve the immune activity of vector vaccine, Improve yield, optimize the effectiveness of build strategies

Inactive Publication Date: 2020-09-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Pseudorabies virus (PRV), porcine epidemic diarrhea virus (Porcineepidemic diarrhea virus, PEDV), porcine acute diarrhea syndrome coronavirus (Swine Acute Diarrhea Syndrome Coronavirus, SADS-CoV), porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome, PRRSV) are common porcine-derived viruses, and a variety of attenuated live vaccines based on these viruses other than SADS-CoV have been developed. However, weakened virulence often leads to reduced immune protection of vaccines. Control of common swine diseases remains limited
[0005] A bottleneck that limits the protective efficacy of vaccines at this stage is that the balance between attenuation and immunogenicity has not been found. Therefore, it is necessary to find a relatively effective replication method for maintaining vaccine strains in pigs to achieve a balance between attenuation and immunogenicity. best balance

Method used

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  • Application of SVA 3C protein in promotion of porcine virus replication
  • Application of SVA 3C protein in promotion of porcine virus replication
  • Application of SVA 3C protein in promotion of porcine virus replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Effect of overexpressing SVA 3C protein on PRV replication

[0042] When the monolayer of PK-15 cells is 80% full, refer to Lipofectamine TM 3000 transfection reagent instructions, the experimental group was transfected with PcDNA3.1-3HA-SVA-3C plasmid, the control group was transfected with PcDNA3.1-3HA empty plasmid, PRV was inoculated with MOI 0.01 after 24 hours, and frozen-thawed samples were collected 24 hours after inoculation. Virus DNA was extracted from the virus liquid, and the copy number of PRV gD gene was detected by fluorescent quantitative PCR. At the same time, the cell extract protein was collected to detect the expression of SVA 3C protein by Western blot, and the virus titer of the two groups of virus liquid was measured. The primers used were as follows:

[0043] PRV gD-F: 5'-CGACTTCATGGTGGCGCTC-3';

[0044] PRV gD-R: 5'-CGCCGAACTTGTACGTGCG-3'.

[0045] In order to explore whether SVA 3C protein can promote PRV replication, the present...

Embodiment 2

[0047] Example 2 Effect of overexpressing SVA 3C protein on replication of PRV attenuated vaccine strain Bartha-K61

[0048] When the monolayer of PK-15 cells is 80% full, refer to Lipofectamine TM 3000 transfection reagent instructions, the experimental group was transfected with PcDNA3.1-3HA-SVA-3C plasmid, the control group was transfected with PcDNA3.1-3HA empty plasmid, 24h later inoculated with PRV Bartha-K61 at MOI 0.01, collected 24h after inoculation The virus DNA was extracted from the freeze-thawed virus solution, and the copy number of PRV gD gene was detected by fluorescent quantitative PCR. At the same time, the protein extracted from the cells was collected to detect the expression of SVA 3C protein by Western blot, and the virus titer of the two groups of virus solutions was measured. The primers used are the same as in Example 1.

[0049] In order to explore whether SVA 3C protein can promote PRV replication, the present invention transfected PcDNA3.1-3HA-SV...

Embodiment 3

[0051] Example 3 Effect of overexpressing SVA 3C protein on PEDV replication

[0052] When the monolayer of A-vero cells is 80% full, refer to Lipofectamine TM 3000 transfection reagent instructions, the experimental group was transfected with PcDNA3.1-3HA-SVA-3C plasmid, the control group was transfected with PcDNA3.1-3HA empty plasmid, PEDV was inoculated with MOI 0.05 after 24 hours, and frozen-thawed samples were collected 24 hours after inoculation. Viral RNA was extracted from the virus liquid and reverse-transcribed into cDNA. The copy number of PEDV N gene was detected by fluorescence quantification. At the same time, the cell extract protein was collected to detect whether the SVA 3C protein was expressed by Western blot. In addition, the virus titer of the two groups of virus liquid was measured. The primers used were as follows:

[0053] PEDV N-F: 5'-TCTACTACCTCGGAACA-3';

[0054] PEDV N-R: 5'-GCAACCCAGAAAACAC-3'.

[0055] In order to explore whether the SVA 3C ...

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Abstract

The invention belongs to the technical field of biology, and experimental research finds for the first time that a protein encoded by an SVA 3C gene can be used as a virus replication enhancer to significantly promote replication of swine viruses including attenuated vaccine strains and inactivated vaccine strains. Therefore, the problems of low virus yield and low vaccine immunogenicity in production and practice at present are well solved. The invention not only provides a new direction for revealing a virus mixed infection mechanism, but also can apply the SVA 3C protein to improve the yield of viruses in production practice, and is more beneficial to optimizing the construction strategies of related genetic engineering live vaccines and live virus vaccine vectors and improving the immune activity of the vector vaccines.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of SVA 3C protein in promoting the replication of porcine virus. Background technique [0002] Pig farming has always been one of the leading industries in my country's agricultural economy. The growing domestic demand for pork consumption and the new standards for food safety and environmental protection have pushed my country's pig farming industry into a highly intensive farming model that is conducive to standardized management. But what followed was enormous pressure on the prevention and control of pig diseases. [0003] Porcine Senecavirus A (Senecavirus A, SVA) is a non-enveloped single-stranded positive-sense RNA virus, which belongs to the Picornaviridae family and is the only member of the Senecavirus genus. The genome length of SVA is about 7.2kb, encoding four structural proteins (VP1, VP2, VP3, VP4) and eight nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N7/00A61K39/245A61P31/14A61P31/22
CPCA61K39/12A61K2039/5252A61K2039/5254A61P31/14A61P31/22C07K14/005C12N7/00C12N2710/16734C12N2710/16751C12N2770/10034C12N2770/10051C12N2770/20034C12N2770/20051C12N2770/32022
Inventor 马静云孙媛李倩妞蓝天陈雨琪
Owner SOUTH CHINA AGRI UNIV
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