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
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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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