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A kind of preparation method of immune-enhanced recombinant prrsv virus-like particle subunit vaccine

A subunit vaccine and immune-enhancing technology are applied in the field of preparation of immune-enhanced recombinant PRRSV virus-like particle subunit vaccine to achieve the effects of good protection of antibodies, high immune protection rate and improvement of antibody levels

Active Publication Date: 2019-10-25
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.

Method used

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  • A kind of preparation method of immune-enhanced recombinant prrsv virus-like particle subunit vaccine
  • A kind of preparation method of immune-enhanced recombinant prrsv virus-like particle subunit vaccine
  • A kind of preparation method of immune-enhanced recombinant prrsv virus-like particle subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0042] Based on the entire gene sequence of the PRRSV strains circulating in the field from 2006 to 2016, using software such as DNAMAN and MEGA5.1 for comparison and analysis, screening the gene sequences of the dominant strains in the field, after artificial modification and codon optimization, send gene synthesis The company conducts gene sequence synthesis and obtains 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 vector pBAC5 were digested with Xba I / BamHI respectively. After recovery and purification, they were ligated with T4 ligase and transformed into DH5α competent cells chemically. After screening positive clones, the plasmid was extracted. Throu...

Embodiment 2

[0068] Example 2 Preparation of immune adjuvant

[0069] The adjuvant is a composite immune adjuvant in the invention patent CN 2017100589467 (CN 106798920 B, authorization announcement date: October 11, 2018) previously applied by the inventor, and after optimization of the composition according to the characteristics of the subunit vaccine The specific formula and preparation method are as follows: a composite immune adjuvant, which is composed of the following components by weight percentage: propolis extract 1%, houttuynia cordata polysaccharide 1.5%, alanine 0.5%, Ginkgo biloba flavone 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 the compound immune adjuvant includes the following steps:

[0071] Step 1: Weigh each raw material according to the weight percentage ratio;

[0072] Step 2: Mix the weighed propolis extract, houttuynia cordata polysaccharid...

Embodiment 3

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

[0083] The recombinant baculovirus Ac-PRRSVGP5-M was inoculated with healthy sf9 cells at a ratio of 5-10%. After culturing at 27°C for 4-5 days, the cells and supernatant were collected by repeated freezing and thawing, and centrifuged at 12000r / min at 4°C for 20 minutes. Collect the supernatant, and then use the ammonium sulfate precipitation method to precipitate the target protein. After resuspension, use diethylene imine (BEI) to inactivate the protein solution for 36-48 hours, and then neutralize with the same amount of sodium thiosulfate, using 0.9 % Normal saline was adjusted to a protein content of 100 μg / mL, and finally mixed with the adjuvant prepared in Example 2 to emulsify a vaccine, and placed at 2-8°C for use.

[0084] During the vaccine preparation process, the recombinant baculoviruses of Example 1, Control 1 and Control 2 of the present invention were cultured and ad...

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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 specifically relates to a method for preparing an immune-enhanced 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), because it often causes cyanosis in the ears clinically. It is called blue-ear disease, which mainly causes premature birth, miscarriage, stillbirth or mummification of sows, and respiratory diseases in piglets and growing pigs, with high morbidity and mortality. The disease was first reported in the United States in the 1980s, and the first clinical virus isolation report was found in my country in 1996. A large-scale outbreak of highly pathogenic PRRSV occurred at the end of 2005. PRRSV is a single-stranded RNA virus belonging to the Arteriviridae family. According to differe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/866C07K14/08A61K39/12A61P31/14
CPCA61K39/12A61K2039/5258A61K2039/552A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2770/10022C12N2770/10023C12N2770/10034
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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