Preparation method of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine

A subunit vaccine and immune enhancement technology, which is applied in the field of preparation of immunoenhanced recombinant PRRSV virus-like particle subunit vaccine, to achieve the effect of good protection antibody, high immune protection rate, and improvement of animal protection rate

Active Publication Date: 2019-03-01
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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  • Preparation method of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine
  • Preparation method of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine
  • Preparation method of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Construction of recombinant baculovirus Ac-PRRSVGP5-M expressing artificially modified and synthesized PRRSV GP5 and M protein

[0041] 1. Synthesis of PRRSV GP5 and M gene sequences:

[0042] 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.

[0043] 2. Construction and identification of recombinant transfer vector:

[0044] 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

[0068] Example 2 Preparation of immune adjuvant

[0069] The adjuvant is based on the compound immune adjuvant in the invention patent CN 2017100589467 (CN 106798920 B, authorized announcement date: October 11, 2018) previously applied by the inventor, and the composition is optimized according to the characteristics of the subunit vaccine Obtained, its specific formula and preparation method are as follows: a kind of composite immune adjuvant, described composite immune adjuvant is made up of the following components of weight percent: propolis extract 1%, houttuynia cordata polysaccharide 1.5%, alanine 0.5%, ginkgo leaf flavonoids 1.5%, polyethylene castor oil 7%, Span80 7%, polyethylene glycol 3.5%, squalene 8%, soybean oil for injection 40%, water for injection 30%;

[0070] The preparation method of described composite immune adjuvant comprises the steps:

[0071] Step 1, each raw material is weighed according to the weight percentage ratio;

[0072] Step 2, mixing the ...

Embodiment 3

[0082] (1) Preparation of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine

[0083] Inoculate healthy sf9 cells with recombinant baculovirus Ac-PRRSVGP5-M at a ratio of 5-10%, culture at 27°C for 4-5 days, collect cells and supernatant by repeated freezing and thawing, and centrifuge at 12000r / min for 20 minutes at 4°C Collect the supernatant, and then use the ammonium sulfate precipitation method to precipitate the target protein. After resuspending, use binary ethyleneimine (BEI) to inactivate the protein solution for 36-48 hours, and then neutralize it with an equal amount of sodium thiosulfate, using 0.9 % normal saline to adjust the protein content to 100 μg / mL, and finally mixed with the adjuvant prepared in Example 2 to emulsify the prepared vaccine and store it at 2-8°C for later use.

[0084] In the vaccine preparation process, after the recombinant baculoviruses of Example 1 of the present invention, control group 1 and control group 2 were cultu...

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Abstract

The invention discloses a preparation method of an immune-enhanced recombinant PRRSV virus-like particle subunit vaccine. The subunit vaccine is prepared from PRRSV virus-like particles and compound immunological adjuvants. By a genetic engineering means, a PRRSV GP5-M gene is modified, and PRRSV virus-like particles with high immunogenicity are prepared by constructing a rhabdovirus expression vector. In addition, by improving the immunological adjuvants, the immuno-enhanced recombinant PRRSV virus-like particle subunit vaccine is obtained, and the subunit vaccine has better immunization effects.

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and in particular relates to a preparation method of an immune-enhancing recombinant PRRSV virus-like particle subunit vaccine. 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. According to the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/866C07K14/08A61K39/12A61P31/14
Inventor 陈瑞杜恩岐董剑辉
Owner 陕西诺威利华生物科技有限公司
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