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Recombinant PRRSV virus-like particles having immunogenicity and preparation thereof

A recombinant baculovirus, virus-like technology, applied in the direction of positive-sense single-stranded RNA virus, medical preparations containing active ingredients, viruses, etc., to achieve the effect of improving immunogenicity, improving animal protection rate, and protecting antibodies

Active Publication Date: 2019-02-26
陕西诺威利华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no vaccine that can completely rid the pig industry of the damage of PRRSV to pigs. The market urgently needs a safer and more efficient PRRS vaccine, and new genetically engineered vaccines are the future development direction of animal vaccines. In 2010, Nam et al published Virus-like particles containing PPRSV GP5 and M proteins have been obtained. The above two proteins are expressed by recombinant baculovirus and then assembled into virus-like particles, which can be used to prepare vaccines. An indispensable part. In 2011, Lu Fenglin et al. cloned the genes encoding M protein, N protein and GP5 protein of PRRSV into eukaryotic expression vectors (such as pHWD2000, pOPI3CAT, pCAGGS, pcDNA6 / TR, pCMV-HA) and simultaneously Transfection of the same cell line to express three proteins can form virus-like particles, and can produce good cellular immunity and humoral immunity. In 2012, Li Yang introduced the two genes ORF5 and ORF6 of PRRSV or the three genes ORF5, ORF6 and ORF7 or ORF5, The four genes ORF6, ORF7 and NSP2a can be combined to form virus-like particles in the baculovirus expression vector. In 2016, Rodriguez et al expressed the GP2\GP3\GP4\E\GP5\M of PRRSV to prepare virus-like particles

Method used

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  • Recombinant PRRSV virus-like particles having immunogenicity and preparation thereof
  • Recombinant PRRSV virus-like particles having immunogenicity and preparation thereof
  • Recombinant PRRSV virus-like particles having immunogenicity and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of recombinant baculovirus Ac-PRRSVGP5-M expressing artificially modified and synthesized PRRSV GP5 and M protein

[0030] 1. Synthesis of PRRSV GP5 and M gene sequences:

[0031] Based on the complete gene sequence of PRRSV virus strains prevalent in the field from 2006 to 2016, use DNAMAN, MEGA5.1 and other software for comparison and analysis, and screen the gene sequences of dominant strains that are prevalent in the field. After artificial modification and codon optimization, send gene synthesis The company synthesized the gene sequence and obtained the tandem sequence GP5-M of GP5 and M.

[0032] 2. Construction and identification of recombinant transfer vector:

[0033] The plasmid containing the target gene sequence GP5-M and the carrier pBAC5 were subjected to Xba I / BamHI double enzyme digestion respectively. After recovery and purification, T4 ligase was used for ligation reaction, chemically transformed into DH5α competent cells, and the plasmid...

Embodiment 2

[0058] (1) Preparation of recombinant porcine reproductive and respiratory syndrome virus virus-like particle vaccine:

[0059] The recombinant baculovirus Ac-PRRSVGP5-M prepared in Example 1 was inoculated into healthy sf9 cells at a ratio of 10%, cultured at 27°C for 4-5 days, and the cells and supernatant were collected by repeated freezing and thawing. Collect the supernatant after centrifugation for 20 minutes, then use the ammonium sulfate precipitation method to precipitate the target protein, resuspend and inactivate the protein solution for 36-48 hours with binary ethyleneimine (BEI), and then use an equal amount of sodium thiosulfate And, finally mixed with Seppic ISA 206 adjuvant to emulsify the prepared vaccine, and put it at 2-8°C for later use.

[0060] In the vaccine preparation process, after the recombinant baculoviruses of Example 1 of the present invention, control group 1 and control group 2 were cultured, they were all adjusted to a virus titer of 10 5 TC...

Embodiment 3

[0070] Embodiment 3: Vaccine safety experiment

[0071] (1) Piglet safety test:

[0072] Randomly select 5 batches of the virus-like particle vaccine prepared in Example 2 prepared in the laboratory, take 3 bottles at random from each batch, mix them uniformly, and inoculate 30-day-old piglets by intramuscular injection with 2 times the dose, and set up a blank control group test at the same time pig. During 3 days before inoculation and 14 days after inoculation, the body temperature of all piglets was measured twice a day, and the spirit and appetite of the experimental pigs were observed. The results showed that the body temperature, mental state and appetite of the test pigs in the immunized group and the control group were normal. It shows that the virus-like particle vaccine is safe for piglets.

[0073] (2) Safety test on pregnant sows

[0074] 5 batches of virus-like particle vaccines prepared in the laboratory were randomly selected, and 3 bottles were randomly se...

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Abstract

The invention discloses recombinant porcine reproductive and respiratory syndrome virus (PRRSV) virus-like particles (VLP) and a preparation method and an application thereof. Based on comparative analysis of GP5 of a PRRSV epidemic strain and an M gene sequence, a GP5 and M tandem sequence GP5M is synthesized artificially, the synthesized GP5M gene sequence is cloned into a vector with a pBAC5 plasmid as a skeleton, the baculovirus transfer vector pBAC-PRRSVGP5M is obtained, the recombinant bacmid rBacmid-GP5M is obtained, sf9 cells are transfected with the bacmid, and the recombinant baculovirus Ac-PRRSVGP5M is obtained. The PRRSV GP5 and M protein are expressed efficiently by the recombinant baculovirus, and the virus-like particles are formed. A subunit vaccine prepared by the proteinexpressed by the recombinant baculovirus can induce a body to produce a specific immune response after immunizing animals and can protect the pig body against the strong poison attacking of porcine reproductive and respiratory syndrome virus.

Description

technical field [0001] The invention belongs to the field of agricultural and veterinary biotechnology, and specifically relates to a recombinant PRRSV virus-like particle with immunogenicity and its preparation and application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV). It is called blue ear disease, which mainly causes premature birth, miscarriage, stillbirth or mummified fetus in sows, and respiratory diseases in piglets and finishing pigs, with high morbidity and mortality. The disease was first reported in the United States in the 1980s, and it was first reported in 1996. The first clinical virus isolation report was found in my country in 2005, and a large-scale outbreak of highly pathogenic PRRSV occurred at the end of 2005. PRRSV is a single-strand positive-strand RNA virus belonging to the family Arteriviridae. Acco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C07K14/08C12N15/866C12N7/01A61K39/12A61P31/14
CPCA61K39/12A61K2039/5258A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2710/14052C12N2770/10022C12N2770/10023C12N2770/10034
Inventor 陈瑞杜恩岐董剑辉张满义
Owner 陕西诺威利华生物科技有限公司
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