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

Inactive Publication Date: 2009-07-29
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inactivated vaccines require a certain preparation process, and in this process, part of the antigen structure is usually destroyed, thus affecting the immune effect
Relatively speaking, protein-based recombinant subunit vaccines h

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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PUM

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Abstract

The invention relates to the field of gene engineering and immunology, in particular to a method for constructing and applying a cross-protective vaccine of Vibrio harveyi and Edwardsiella tarda, and an antigen expression vector thereof. The cross-protective vaccine has a base sequence in a sequence table SEQ ID No.1, and is constructed by the following steps: constructing plasmids pPT6 and pT6VH; and on the basis, constructing a plasmid pT6VH18 containing the antigen of the cross-protective vaccine of Vibrio harveyi and Edwardsiella tarda. The pT6VH18 is converted into colibacillus to be cultured under a conventional condition so as to obtain the protective vaccine. The vaccine not only has cross-protection effect, but also does not need any purifying process.

Description

technical field [0001] The invention relates to the fields of genetic engineering and immunology, in particular to a cross-protection vaccine of Vibrio harveyi and Edwardsiella lentus and a construction method thereof. Background technique [0002] Vibrio harveyi and Edwardsiella tarda are important pathogens of aquaculture organisms in the world, including my country, both of which have a wide range of hosts and can infect a variety of aquaculture animals, such as fish, shrimp, and shellfish. In addition, Edwardsiella tarda can still infect humans and is a zoonotic bacterium. At present, the prevention and treatment of Vibrio harveyi and Edwardsiella mainly rely on antibiotics (including various chemicals, pesticides, etc.) and traditional vaccine immunization, that is, simple inactivated vaccines. Although there are research reports on recombinant subunit vaccines against these two pathogens, they have not been seen in practical applications. Inactivated vaccines require...

Claims

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Application Information

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IPC IPC(8): A61K39/106A61P31/04C12N15/63C12N15/62C12N15/31A61K39/02
Inventor 孙黎孙鲲
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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