A kind of recombinant prrsv virus-like particle with immunogenicity and its preparation
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 high immune protection rate, good protection of antibodies, and improvement of animal protection rate
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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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