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Edwardsiella tarda recombinant subunit vaccine and application thereof

A delayed Edwards, subunit vaccine technology, applied in the fields of molecular biology and immunology, to achieve high protection rate and simple application effect

Inactive Publication Date: 2011-03-30
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although there are several cases of Edwardsiella tarda recombinant subunit vaccine antigen reports at present, there is only one case where the protective effect (relative survival rate) reaches more than 70% (reported by Japanese scientific researchers)
Therefore, recombinant subunit vaccines with high protective effects have yet to be developed

Method used

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  • Edwardsiella tarda recombinant subunit vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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...

Embodiment 2

[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...

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Abstract

The invention relates to the field of molecular biology and immunology and particularly discloses an Edwardsiella tarda recombinant subunit vaccine as well as a preparation method and application thereof. The vaccine has a base sequence in a sequence table SEQ ID No.1. The preparation method of the Edwardsiella tarda recombinant subunit vaccine comprises the following steps of constructing a vaccine antigen expression plasmid pETXV21, converting the vaccine antigen expression plasmid pETXV21 into escherichia coli BL21 (DE3), recovering a recombinant protein after induced expression, mixing the recombinant protein with a strain B187, and dissolving the mixture in PBS (Phosphate Buffer Saline) to obtain a vaccine mixed liquor which has the function of immune protection to Edwardsiella tarda. The vaccine mixed liquor is intraperitoneally injected to fishes to achieve the purpose of immune protection.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to an Edwardsiella tarda recombinant subunit vaccine and its preparation and application. Background technique [0002] Edwardsiella tarda is a Gram-negative pathogen with a broad host range, including humans, animals and fish. In my country, Edwardsiella tarda is an important pathogen of a variety of cultured fish (turboscus, flounder and eel, etc.), and the Edwardsiella disease induced by it causes huge economic losses to the aquaculture industry every year. Prevention and control of tardive Edwardsiella currently relies on the use of antibiotics and vaccines, the latter of which is limited to a small number of countries. Due to the well-known hazards of antibiotics, their use has gradually been limited. Contrary to antibiotics, vaccines are recognized as one of the most comprehensive disease prevention measures. Common vaccines include inactivated vaccines, sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61P31/04C12N15/70C12N15/31C12P21/02C12R1/19
Inventor 孙黎焦绪栋
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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