Porcine reproductive and respiratory syndrome chimeric virus rvhbjx and its application

A respiratory syndrome, chimeric virus technology, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the problem that the in vivo test has not been carried out, and achieve improved immune protection efficacy, good safety, genetic characteristics stable effect

Active Publication Date: 2019-05-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Porcine reproductive and respiratory syndrome chimeric virus rvhbjx and its application
  • Porcine reproductive and respiratory syndrome chimeric virus rvhbjx and its application
  • Porcine reproductive and respiratory syndrome chimeric virus rvhbjx and its application

Examples

Experimental program
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Effect test

Embodiment 1

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

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

[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 in the HB-1 / 3.9 gen...

Embodiment 2

[0056] Example 2 Rescue of the chimeric virus RvHBJX

[0057] Take 15 μg of the pW-HJSP full-length cDNA cloning plasmid prepared in Example 1, digest it with Rsr II, Pac I (NEB, Beijing, China) endonuclease, and bathe it overnight at 37° C. to completely linearize the closed loop plasmid. Add phenol chloroform for extraction 2-3 times, precipitation with absolute ethanol, after centrifugation and drying, add 10μL of RNase-free water (Ambion, Texas, USA) to dissolve the precipitate, and measure the purified plasmid concentration by UV spectrophotometer. Use Ambion’s High Yield Capped RNATranscription kit for in vitro transcription. After reacting in a 37°C water bath for 2 hours, add 1μL of TURBO DNase I and mix well. After a brief centrifugation, incubate at 37°C for 15 minutes to digest the template plasmid DNA.

[0058] Select MARC-145 cells with normal morphology and vigorous growth. After trypsinization, 6 The cell density of cells / mL was transferred to a six-well cell cult...

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 virus 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 generation viruses were respectively inoculated into MARC-145 cells, and immunofluorescence test was performed with PRRSV N protein monoclonal antibody to observe the expression of 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. The control cells have no visible fluorescence ( image 3 ). It shows that the inserted structural protein can be expressed correctly 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 its application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), mainly caused by abortion, premature delivery, stillbirth and mummies in pregnant sows Fetal and other reproductive disorders are characterized by respiratory symptoms in piglets and growing-finishing pigs (Rossow et al., 1994). Since the early summer of 2006, severe PRRS outbreaks have quickly swept across my country's major pig-raising areas, causing a large number of pig deaths and causing huge economic losses to the pig industry in my country. Vaccines are considered to be an important tool for the prevention, control and purification of PRRSV. However, PRRSV infection can cause immunos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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