Cross-protection vaccine and building method thereof
A cross-protection and construction method technology, applied in the fields of genetic engineering and immunology, can solve the problems of antigen structure destruction, expensive preparation, purification of large proteins, etc., and achieve high protection rate, simple application, and simple preparation process
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Embodiment 1
[0023] The protective vaccine antigen of the present invention is shown by the base sequence of vhe18 in the sequence table SEQ ID No.1.
[0024] The construction method of vaccine antigen surface display vector:
[0025] 1) Construction of plasmid pPT6
[0026] Using the plasmid pTrcHis (purchased from Invitrogen, USA) as a template, the following primers were used for PCR: TRF1 (5'- GAATTC ATTAATCACTGCATAATTCGTG-3', the underlined base is the EcoRI site), TRR1 (5'- GGATCCGATATC ATTATACGAGCCGGATG-3', the underlined base is the BamHI / EcoRV site), the PCR conditions are: 94°C 60s pre-denatured template DNA, then 94°C 40s, 48°C 60s, 72°C 60s, after 5 cycles, change to 94°C 40s, 57°C for 60s, 72°C for 60s, 25 cycles and then extended reaction at 72°C for 10min. After the PCR product was purified with Tiangen DNA Product Purification Kit, it was ligated with the PCR cloning vector pBS-T (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) at room temperature fo...
Embodiment 2
[0056] Immunization Application of Vaccine
[0057] Step 1) Immunization injection of vaccine. Suspend the collected cells in PBS to a final concentration of 5×10 8 cfu / ml is the vaccine preparation solution. 120 flounder (each weighing about 13 g) were randomly divided into 4 groups, 30 in each group. Name these 4 groups as Groups A, B, C, and D, respectively. Each fish in groups A and C was injected intraperitoneally with 100 ul of the above-mentioned vaccine preparation solution. Each fish in groups B and D was injected intraperitoneally with 100ul PBS respectively.
[0058] The composition of PBS is: 0.8g NaCl, 0.02g KCl, 0.358g Na 2 HPO 4 .12H 2 O, 0.024g NaH 2 PO 4 .
[0059] Step 2) Preparation of suspensions of Vibrio harveyi and Edwardsiella tarda. Culture Vibrio harveyi T4 and Edwardsiella tarda TX1 to OD separately in LB medium 600 0.6, and then centrifuged (5000g, 4°C) for 10min. Collect the cells and suspend them in PBS to a final concentration of 5×1...
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