Chimeric virus RvHBJX of porcine reproductive and respiratory syndrome and application of chimeric virus

A technology of respiratory syndrome and chimeric virus, which is applied in the direction of antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve the problems that the in vivo test has not yet been carried out, and achieve the improvement of immune protection effect, good safety, and genetic characteristics stable effect

Active Publication Date: 2015-05-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Lee et al. replaced the corresponding region in the infectious cDNA clone plasmid with the structural protein coding region of the Korean PRRSV epidemic strain and rescued the corresponding PRRSV chimeric virus, which can be neutralized by the serum of naturally infected pigs, and the corresponding In vivo testing has not yet been performed

Method used

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  • Chimeric virus RvHBJX of porcine reproductive and respiratory syndrome and application of chimeric virus
  • Chimeric virus RvHBJX of porcine reproductive and respiratory syndrome and application of chimeric virus
  • Chimeric virus RvHBJX of porcine reproductive and respiratory syndrome and application of chimeric virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of chimeric virus full-length cDNA cloning plasmid

[0049] Referring to the complete genome sequence of PRRSV HB-1 / 3.9 (GenBank accession number EU360130) and PRRSV JXwn06 whole genome sequence (GenBank accession number EF641008), primers were designed for the construction of PRRSV chimeric strain RvHBJX full-length cDNA clone (Table 1), The primers were synthesized by American Invitrogen Yingjie Life Technology Co., Ltd. (Invitrogen, Beijing, China), and the concentration was prepared with nuclease-free water.

[0050] Table 1 Primers for HBJX full-length cDNA clone construction

[0051]

[0052]

[0053] Note: The horizontal line is the restriction endonuclease recognition sequence (including the protective bases on both sides to increase the efficiency of digestion); the primer sequence is written in capital letters, indicating that HB-1 / 3.9 is used as a template for PCR amplification , the corresponding position represents the position...

Embodiment 2

[0056] Example 2 Rescue of chimeric virus RvHBJX

[0057] Take 15 μg of the pW-HJSP full-length cDNA cloning plasmid prepared in Example 1, digest with Rsr II and Pac I (NEB, Beijing, China) endonucleases, and bathe overnight at 37° C. to completely linearize the closed-loop plasmid. Add phenol chloroform for extraction 2-3 times, precipitate with absolute ethanol, centrifuge and dry, add 10 μL RNase-free water (Ambion, Texas, USA) to dissolve the precipitate, and measure the concentration of the purified plasmid with an ultraviolet spectrophotometer. Immediately use Ambion's High Yield Capped RNA Transcription kit for in vitro transcription. After reacting in a water bath at 37°C for 2 hours, add 1 μL of TURBO DNase I to mix well, centrifuge briefly and incubate at 37°C for 15 minutes to digest the template plasmid DNA.

[0058] Select MARC-145 cells with normal morphology and vigorous growth, and digest them with trypsin for about 10 6 The cell density per mL was transfe...

Embodiment 3

[0060] Example 3 Expression of JXwn06 structural protein in chimeric strain RvHBJX

[0061] The gene sequence determination of the third-generation chimeric virus RvHBJX showed that its structural protein coding region was consistent with that of JXwn06, and no gene deletion or mutation occurred.

[0062] N protein is a non-glycosylated structural protein encoded by ORF7 of PRRSV genome. RvHBJX, RvHB-1 / 3.9, and RvJXwn P3 viruses were inoculated into MARC-145 cells respectively, and immunofluorescence experiments were performed with PRRSV N protein monoclonal antibody to observe the expression of the replacement protein in the chimeric virus. Bright yellow-green fluorescence can be seen in the cytoplasm infected by the chimeric virus RvHBJX, which is the same as the parental rescue virus RvHB-1 / 3.9 and RvJXwn, and the control cells have no visible fluorescence ( image 3 ). It shows that the inserted structural protein can be correctly expressed in the chimeric strain.

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Abstract

The invention provides a chimeric virus RvHBJX of porcine reproductive and respiratory syndrome and an application of the chimeric virus and belongs to the technical field of genetic engineering in virus. The chimeric virus RvHBJX of porcine reproductive and respiratory syndrome provided by the invention is a chimeric virus which comprises a JXwn06 structural protein coding region of a high pathogenicity PRRSV strain by taking a low pathogenicity PRRSV strain HB-1 / 3.9 as a framework. After the chimeric virus is continuously passed for 80 times in vitro through an MARC-145 cell, the hereditary property of the virus is stable. The adaptability of the virus on a host cell is gradually enhanced along increase of the passage numbers. After the chimeric virus is passed for 60 times (P60) or over 60 times, the animal body is relatively good in safety, and the inoculated virus can provide 100% immune protection for high pathogenicity PRRSV strain and can be used as a vaccine candidate for the porcine reproductive and respiratory syndrome. The chimeric virus for preventing and treating porcine reproductive and respiratory syndrome has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of viral genetic engineering, in particular to a porcine reproductive and respiratory syndrome chimeric virus RvHBJX and applications thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), mainly causing abortion, premature birth, stillbirth and mummification in pregnant sows Fetuses and other reproductive disorders and respiratory symptoms in piglets and growing-finishing pigs are characterized (Rossow et al., 1994). Since the early summer of 2006, severe PRRS broke out and quickly swept through the main pig-raising areas in my country, resulting in the death of a large number of pigs and causing huge economic losses to the pig-raising industry in my country. Vaccines are considered to be an important tool for the preventio...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C12N15/66A61K39/12A61P31/14C12R1/93
Inventor 盖新娜杨汉春郭鑫周磊张家龙颜忠姚晟晨
Owner CHINA AGRI UNIV
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