Edwardsiella tarda recombinant protein vaccines, and preparation and application thereof

A technology of Edwards tarda and recombinant protein, applied in the field of Edwards tarda recombinant protein vaccine and its preparation, can solve the problems of industrial economic loss, Edwardian tarda vaccine, late start and other problems, and achieve high protective effect

Inactive Publication Date: 2012-08-01
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the aquaculture industry, Edwardsiella tarda is an important fish pathogen that has been reported to cause disease in a variety of marine and freshwater fish, causing severe economic losses to the industry
At present, Sout

Method used

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  • Edwardsiella tarda recombinant protein vaccines, and preparation and application thereof
  • Edwardsiella tarda recombinant protein vaccines, and preparation and application thereof
  • Edwardsiella tarda recombinant protein vaccines, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The two vaccine antigens of Edwardsiella tarda are shown in the amino acid sequences of SEQ ID No. 1 and SEQ ID No. 2 in the sequence table (see sequence table 1 and 2).

[0024] Sequence listing SEQ ID No.1

[0025] MENNLLGDGFKLPKGLTDYGAAAQSWNQYAEKNGLTPEQKQAGLDRLAKGDLPEDTNITKAIVEGYQDGVMIAETWYLGP

[0026] AASVRKVIGGGIIAEIANGSYQWFDLSQPGNGNKSWDWKSSTSAGITGILAPRRSIGQNVGIAMGSAFFTDGPDTGSIGG

[0027] AAAGAWAGGLFGEYASGIVNSLTSKKVPGFIFNTTGSFSSEILGGYIKDAVNGTQLSSERNKEVGE

[0028] (a) Sequence features:

[0029] ●Length: 226

[0030] ●Type: amino acid sequence

[0031] ●Chain type: single chain

[0032] ●Topological structure: linear

[0033] (b) Molecule type: protein

[0034] (c) Assumption: No

[0035] (d) Antisense: No

[0036] (e) Original source: Edwardsiella tarda TX1

[0037] (f) Specific name: E226

[0038] Sequence listing SEQ ID No.2

[0039] ASEKTMDRPGNLIKENQQRAASDEVGRLFRMPITPDGTAVLSGEIGQQPIAIHNVEDANAGELVDSPINDAIAININRASQNNKNN

[0040] AGAGSLTKEQDPMDSLSIRGVG...

Embodiment 2

[0056] The construction method of Edwardsiella tarda vaccine expression vector:

[0057]1) Construction of plasmid pE226: use Edwardsiella tarda TX1 as a template, and use F1 / R1 as primers for PCR amplification. The PCR conditions are: 94°C for 60s to pre-denature the template DNA, then 94°C for 40s, 55°C for 60s, and 72°C 60s, 30 cycles and then extended reaction at 72°C for 10min. After the PCR product was purified with the Tiangen DNA Product Purification Kit, it was ligated with the vector pEASY-E2 (purchased from "Beijing Quanshijin Biotechnology Co., Ltd.") at room temperature for 2-4 hours, and the ligation mixture was transformed into Escherichia coli DH5α containing 100ug / ml Anka penicillin (Ap) was cultured on LB solid medium for 18 hours, a transformant was picked, and the plasmid was extracted, which was plasmid pE266.

[0058] The LB composition is calculated by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride, 97.5% distilled water; the b...

Embodiment 3

[0063] Inducible expression and purification of vaccine proteins

[0064] 1) Induced expression and purification of E226 vaccine protein: The plasmid pE226 in the above step 1) was transformed into Escherichia coli BL21 (DE 3) (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) by conventional methods, and the plasmid containing Ap( 100ug / ml) on LB solid medium for 18-24 hours, pick a transformant, and name it BL21 / pE226. Cultivate BL21 / pE226 in LB liquid medium containing Ap (100ug / ml) overnight; take 1ml of overnight culture solution, add 100ml of fresh LB liquid medium containing Ap (100ug / ml), at 37°C Shake at 200rpm to OD 600 0.6, add IPTG with a final concentration of 1mM, continue shaking culture at 37°C at 160rpm for 4-5h, then centrifuge at 5000g, 4°C for 10min, collect the bacterial liquid, add 5ml of lysate, and shake slowly on a shaker at room temperature for 1- 2 hours until the bacterial suspension becomes clear. The bacterial liquid was centr...

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Abstract

The invention relates to the field of molecular vaccinology, in particular to two Edwardsiella tarda recombinant protein vaccines, and a preparation method thereof. The antigens of the vaccines are shown as amino acid sequences of SEQ ID No.1 and SEQ ID No.2 in a sequence table. The vaccines are applied without commercial adjuvants, and can achieve an immune protection effect of over 80 percent.

Description

technical field [0001] The invention relates to the field of molecular vaccinology, in particular to Edwardsiella tarda recombinant protein vaccine and its preparation method. Background technique [0002] Edwardsiella tarda is a Gram-negative pathogen with a broad host range, including humans, fish, and birds. For the aquaculture industry, Edwardsiella tarda is an important fish pathogen. It has been reported to cause diseases in a variety of seawater and freshwater fish, causing serious economic losses to the industry. At present, South Korea has a commercialized vaccine based on inactivated whole bacteria, but in my country, due to the late start of related research, there is no effective Edwardsiella tarda vaccine that can be applied to industry so far. Therefore, the research and development of vaccines against Edwardsiella tarda urgently needs to be carried out in our country. Vaccines come in many different forms, one of which is a recombinant subunit vaccine based ...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61P31/04C12N15/70C12N15/66C12P21/02C12R1/19C12R1/01
Inventor 孙黎李墨非孙云
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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