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PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof

An immunogenic, virus-like technology, applied in the direction of antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve the problems of poor immune efficacy and strong virulence, and achieve high immunogenicity and immune protection rate high effect

Active Publication Date: 2014-02-05
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These include classic PRRS inactivated vaccines and attenuated vaccines, HP-PRRS inactivated vaccines and attenuated vaccines, because live vaccines generally have the problem of strong virulence, and the main problem of inactivated vaccines is poor immune efficacy
There is no satisfactory vaccine in the world for the prevention of this disease. At present, the vaccine purchased by the Chinese government in large quantities is the highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain), which is produced by Highly pathogenic and strong virus strains are made by continuous passage of cells to weaken, and the vaccine also has the risk of returning to strong virulence

Method used

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  • PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof
  • PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof
  • PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction of Baculovirus

[0077] According to the TakaR MiniBEST Viral RNA / DNA (code: DV819A) kit instructions, the highly pathogenic porcine reproductive and respiratory syndrome virus Guangdong isolate RNA was extracted, using oligo(dT) as a primer, and using M-MLV reverse transcriptase (Invitrogen , Carlsbad, CA, USA) for reverse transcription to obtain cDNA.

[0078] Referring to the GenBank accession number: EU109503.1 gene sequence, the upstream primer 5'-GGATCCATGCCAAATAACAACGGCAAGC-3' and the downstream primer 5'-AAGCTTTCATGCTGAGGGTGATGCTGTG-3' with restriction sites were designed to amplify the N protein encoding PRRSV GD strain with cDNA as template The amplified fragment was named as PRRSV-N; it was recovered by double digestion with BamH Ⅰ and Hind Ⅲ, subcloned into the pFastBacDual vector (Invitrogen, Carlsbad, CA, USA) (abbreviated as pFBD) treated with the same digestion, and constructed The pFBD-PRRSV-N recombinant vector was obtained.

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Embodiment 2

[0082] Example 2 Expression of PRRSV VLPs in insect cells

[0083] SF9 insect cells were infected with the Bac-PRRSV-N-GP5 recombinant baculovirus expression vector at the MOI specified by the instructions of Invitrogen. After the recombinant virus infected SF9 cells for 72 hours, the cells were washed twice with PBS, fixed with 80% (v / v) cold acetone for 20 minutes, washed three times with PBS, and the mouse monoclonal antibody against PRRSV N protein or anti-PRRSV GP5 protein antibody (anti-PRRSV GP5 protein) was added. PRRSV GP5 protein (rabbit polyclonal antibody) primary antibody (1:100) 37 ℃ for 1 hour, PBS washed three times, FITC-labeled goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody (1:500) 37 ℃ for 1 hour, PBS wash Three times, the slides were mounted with 50% (v / v) glycerol and observed under a fluorescence microscope.

[0084] The antibody against PRRSV GP5 protein was used to detect the expression of the target protein in infected insec...

Embodiment 3

[0087] Embodiment 3 Immunogenicity of three kinds of PRRSV virus-like particles in mice

[0088] The mice were immunized with the crudely purified three kinds of virus-like particles to evaluate the immunogenicity of the virus-like particles. On the 1st and 14th day, the crudely purified three kinds of virus-like particles were injected subcutaneously with 1 μg / mouse, and the aluminum adjuvant The agent is an adjuvant (ratio of 3:7 to antigen). The control group was immunized with substances expressed by pFBD empty vector without exogenous gene and aluminum adjuvant. The mouse serum was collected 7, 14, 21, and 28 days after immunization, and the N antibody was determined with the PRRSV N antibody detection kit (Table 1) and the GP5 antibody was determined with the PRRSV GP5 antibody detection kit (Table 2), The results showed that the prepared Bac-N-GP5 virus-like particle vaccine immunized group had the highest positive serum N antibody and GP5 antibody on day 21 and day 28...

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Abstract

The invention discloses PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof, and belongs to the technical field of agricultural veterinary biology. The preparation comprises the following steps: cloning coding sequences of N protein and GP5 protein of PRRSV into rod granules to obtain recombinant rod granules; infecting the recombined rod granules on insect cells, culturing the transfected insect cells to obtain recombinant baculovirus. The N protein and GP5 protein are expressed by adopting the recombinant baculovirus infected insect cells, protein with better steric configuration can be obtained, and PRRSV virus-like particles can be automatically assembled. The virus-like particles with high immunogenicity can be used for preparing vaccine for preventing infection of PRRSV. The PRRSV virus-like particles have high immune protective rate, can produce attacking protection on PRRSV virulent strain, can be safer and more effective, and do not have the risk of virulence return.

Description

technical field [0001] The invention belongs to the field of agricultural and veterinary biotechnology, and in particular relates to an immunogenic PRRSV virus-like particle and its preparation and application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is an infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which seriously endangers the pig industry. The disease was first discovered in the United States in 1987. In 1991, the Central Veterinary Research Institute of the Netherlands determined that the pathogen of the disease was a new RNA virus and named it Lelystad virus. In 1996, Guo Baoqing and others isolated PRRSV from domestic suspected PRRS-infected pigs for the first time, thus confirming the existence of the disease in my country. In April 2006, highly pathogenic porcine reproductive and respiratory syndrome broke out in pig herds in my country. Highly pathogenic porcine reproductive a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/12A61P31/14
Inventor 杨雷漆世华王焕君李玉萍谢红玲温文生舒银辉朱薇冯钊
Owner WUHAN CHOPPER BIOLOGY
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