A kind of recombinant pcv2 virus-like particle propolis vaccine and its preparation method
A virus-like, particle-like technology, applied in botany equipment and methods, biochemical equipment and methods, viruses, etc., can solve the problems of inability to stimulate cellular immunity, emergency prevention and treatment, etc., achieve good cellular immunity and offset the loss of slow growth Effect
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Embodiment 1
[0036] Embodiment 1: Construction of recombinant escherichia coli
[0037] 1.1 Sequence optimization and construction of recombinant vector
[0038] According to the determined ORF2 coding sequence in the gene sequence (Genbank accession number: JF272498) of the PCV2 strain isolated from Binzhou Institute of Animal Husbandry and Veterinary Medicine in Shandong Province, the ORF2 encoding Cap protein was optimized to obtain SEQ ID No: 1 Protein sequence, on this basis, according to the codon preference design ORF2 coding sequence SEQ ID No: 2, synthesized by Shanghai Bioengineering Co., Ltd. and construct the prokaryotic expression plasmid pET of PCV-2 Cap protein based on pET-28a(+) -BZCap, specifically insert the coding sequence of ORF2 SEQ ID No: 2 into pET-28a(+) BamHI and Hind III Between the sites, it was verified by double enzyme digestion, and the results are shown in figure 1 .
[0039] Transformation of recombinant plasmid pETBZCap
[0040]Transform the recombi...
Embodiment 2
[0046] Example 2: Induced expression of recombinant bacteria and purification of recombinant protein
[0047] 2.1 High-density fermentation of Escherichia coli without pathogenic endotoxin and high-efficiency expression of Cap protein
[0048] PCV2 Cap protein without pathogenic endotoxin-free Escherichia coli ClearColi TM BL21(DE3)-pET-BZCap was inoculated into 200L high-efficiency expression medium according to the inoculation amount of 2%, and cultured at 37°C until OD 600 When it reaches about 10, add IPTG to a final concentration of 1mmol / L, continue to induce culture for 5 hours, control dissolved oxygen at 30%-50%, and pH at 7.2-7.8.
[0049] Preparation of recombinant protein virus-like particles
[0050] Centrifuge the induced bacterial solution at 10,000r / min for 30 minutes in a tube centrifuge, collect the bacterial cells, wash with PBS (10mmol / L, pH7.2), add PBS buffer to concentrate at a volume ratio of 1 / 10, and use a high-pressure homogenizer Broken, after hi...
Embodiment 3
[0069] Embodiment 3: Preparation of recombinant PCV2 virus-like particle vaccine
[0070] 3.1 Inactivation
[0071] Put the protein solution in the inactivation container, add the formaldehyde solution in a metered amount, and shake it as it is added to make it fully mixed. The final concentration of the formaldehyde solution is 0.2%. Take it out after inactivation at 37°C for 16 hours, and store it at 2-8°C.
[0072] Semi-finished product inspection
[0073] 3.2.1 Sterility test According to the appendix of the current "Chinese Veterinary Pharmacopoeia", the sterility test should be qualified.
[0074] 3.2.2 Determination of protein content The protein content was detected by the Bradford method, and the protein concentration was determined to be ≥ 2.0 mg / ml.
[0075] 3.2.3 The titer of virus-like particle antigen detected by agar amplification method is above 1:32.
[0076] 3.2.4 The non-pathogenic endotoxin content should be ≤10000EU / mL as detected by the Limulus reagent...
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