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pcv2-prrsv recombinant virus and its preparation method, gene, application and vaccine

A PCV2-PRRSV, 1. PCV2-PRRSV technology, applied in the field of viruses, can solve the problems of infection, growth retardation, increased mortality, etc., and achieve the effect of simple operation and easy control

Active Publication Date: 2021-03-19
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Both PRRSV and PCV2 can cause severe diseases in pigs. PRRSV can cause severe reproductive disorders in sows, and common symptoms of pneumonia, growth retardation and increased mortality in weaned pigs; PCV2 is an immunosuppressive piglet that highly infects piglets. The viral infectious disease of pigs can cause pigs of all ages to be infected and cause huge economic losses to the breeding industry

Method used

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  • pcv2-prrsv recombinant virus and its preparation method, gene, application and vaccine
  • pcv2-prrsv recombinant virus and its preparation method, gene, application and vaccine
  • pcv2-prrsv recombinant virus and its preparation method, gene, application and vaccine

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preparation example Construction

[0069] The present invention also provides the above-mentioned PCV2-PRRSV recombinant virus, the gene encoding the above-mentioned PCV2-PRRSV recombinant virus, and the above-mentioned PCV2-PRRSV recombinant virus, or the biological material related to the above-mentioned gene, and the application of the preparation method of the above-mentioned PCV2-PRRSV recombinant virus . The application includes: preparing PCV2 and / or PRRSV antigens or antibodies, the antibodies can optionally be injected into mammals with substances containing PCV2 and / or PRRSV antigens, and then collecting antibodies produced in mammals, or B cells capable of producing antibodies are collected, and then monoclonal antibodies are obtained by preparing hybridoma cells. Correspondingly, the application may also include preparation of reagents and / or kits, reagents and / or kits include but are not limited to: PCV2-PRRSV recombinant virus, gene encoding the above-mentioned PCV2-PRRSV recombinant virus, the ab...

Embodiment 1

[0078] Example 1 PCV2-PRRSV Gene Synthesis

[0079] Obtain PRRSV and PCV2 type genomes according to logging into GenBank, and finally determine the candidate gene sequence through sequence comparison analysis, and the neutralizing epitope gene sequence of PRRSV comes from GenBank: JN654459, and the present embodiment has the PRRSV polypeptide as described in SEQ ID NO.6 For example, the PRRSV polypeptide as described in SEQ ID NO.6 is derived from the Cap5 protein of PRRSV, and the Gp5 protein is an envelope glycoprotein encoded by ORF5 of PRRSV, which is the only glycoprotein in the structural proteins that constitute PRRSV, and is exposed to viral There are epitopes that can interact with the virus on the surface of the particles, and contain at least two neutralizing antigenic determinants, which are the main protective antigens of PRRSV. The PRRSV Gp5 polypeptide gene sequence and the PCV2 Cap protein gene sequence are connected end-to-end through the sequence. Insert res...

Embodiment 2

[0080] Example 2 Construction of pBluescriptⅡPCV2-PRRSV plasmid

[0081] (1) Digestion: Use restriction endonuclease HindⅢ to double digest the positive cloning vector and vector pBluescript SKⅡ(+) containing the target gene respectively. The specific reaction system is as follows:

[0082]

[0083]

[0084] After adding the above components, perform transient centrifugation and mix, 37 ° C for 3 hours, after the reaction is completed, purify and recover the enzyme-cut fragments according to the instructions of the OMEGA plasmid purification and recovery kit. The purified product was dissolved in 30 μL DNase- and RANase-free triple-distilled water.

[0085] (2) Ligation: Carry out ligation reaction in a 200 μL PCR tube according to the instructions of T4 DNA ligase. 10μL connection system is as follows:

[0086] Digested and purified product 2μL pBluescript SKⅡ(+) digested purified product 1μL 10x Ligase Buffer 1μL T4 DNA ligase 1μL ...

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Abstract

The invention provides a PCV2-PRRSV recombinant virus and a preparation method, a gene, an application and a vaccine thereof, and relates to the technical field of virus. The recombinant virus expresses a PRRSV antigen with a PCV2 virus as a vector, and has the advantages of simple operation and easy control by using the characteristic that the PCV2 genome is small and capable of rolling circle replication. The PRRSV antigen is selected from polypeptide with good immunogenicity in PRRSV, and the recombinant virus obtained by inserting the PRRSV antigen into the PCV2 virus has dual antigenic characteristics of PRRSV and PCV2, and can make a body produce immune response to PRRSV and PCV2 at the same time. Based on the concept of the invention, the invention also provides a gene mainly composed of a gene sequence encoding PCV2 and a gene sequence encoding the PRRSV antigen, a biomaterial containing the gene, and a vaccine, wherein the vaccine provided by the invention has diimmunogenicityof PRRSV and PCV2, and can achieve the goal of preventing two diseases with one needle.

Description

technical field [0001] The invention relates to the technical field of viruses, in particular to a PCV2-PRRSV recombinant virus and its preparation method, gene, application and vaccine. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus belonging to the family Arteriviridae and a member of the genus Arteriviridae. In 1991, the Dutch Central Veterinary Research Center took the lead in isolating the virus from artificially and naturally infected pigs, and named the Lelystad virus (LV) American isolate as VR-2332. The PRRSV virion is oval in shape, 50-65nm in diameter, 30-50nm in diameter of the nucleocapsid, and has short Snm protrusions on the capsid. The virus has an envelope, icosahedral symmetry, and a genome length of 15 432b, including 8 open reading frames (ORFs). ORF1 is the largest reading frame, about 12kb long, accounting for about 8000bp of the genome. ORF2-7 is a structural ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/86C12N15/62A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/14C07K14/005C07K2319/00C12N15/86C12N2750/10022C12N2750/10043C12N2770/10022C12N2770/10034
Inventor 贺笋张伟李延涛李俊辉程兰玲高窦潘晓梅孙黎
Owner 天康生物制药有限公司