Edwardsiella tarda recombinant subunit vaccine and application thereof

[0022] The vaccine XV21 is shown by the base sequence in the sequence table SEQ ID No.1.

[0023] The preparation method of vaccine XV21:

[0024] 1) Construction of plasmid pETXV21

[0025] Plasmid pET28 (purchased from Novagen, USA) was digested with NdeI / XhoI, and a 5.3 kb fragment was recovered. With Edwardsiella tarda TX1 (preserved in CGMCC, preservation number is: CGMCC No.2330, preservation certificate has been submitted in application 200810016124.3 Edwardsi tarda vaccine antigen and its preparation method in 20080509) as template, with primer XVF (5 '-GCG CATATG CAATACACCAAATTGCGTT-3', the underlined base is the NdeI site) and XVR (5'- CTCGAG CACCCGGTTATACTCCT-3', the underlined base is the XhoI site) Perform PCR under the following conditions: pre-denatured template DNA at 94°C for 60s, then 94°C for 40s, 53°C for 60s, 72°C for 60s, and after 5 cycles, change to 94°C for 40s , 61°C for 60s, 72°C for 60s, and after 25 cycles, extend the reaction at 72°C for 7-1...

[0042] Immune Application of Recombinant Subunit Vaccine XV21

[0043] Step 1) Preparation of vaccine mixture.

[0044] Bacterial strain B187 (preserved in CGMCC, deposit number: CGMCC No.2331, deposit certificate has been submitted in application 200810014711.9 a cross-protective vaccine antigen and its preparation method and application in 20080229) was cultured in LB medium at 30°C to OD 600 0.8, and then centrifuged (5000g, 4°C) for 10min. Collect the cells and suspend them in PBS to a final concentration of 2x10 8 cfu / ml, namely B187-PBS. Then, 0.2 mg of the vaccine antigen XV21 purified in Step 2 of Example 1 above was added to each milliliter of B187-PBS mixture;

[0045] The composition of PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the rest is water.

[0046] Step 2) Immunization application of the recombinant subunit vaccine XV21. Sixty flounder (each weighing about 10 g) were randomly divided into...