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DNA vaccine for Edwardsiella tarda and construction and application thereof

A technology of Edwards tarda, DNA vaccine, applied in Edwards tarda DNA vaccine and its construction and application fields, to achieve the effect of high protection rate and efficient immune protection

Inactive Publication Date: 2011-06-08
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Edwardsiella tarda DNA vaccine has not been reported so far

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The DNA vaccine is shown by the base sequence in the sequence table SEQ ID No.1.

[0024] Construction and preparation method of DNA vaccine:

[0025] 1) Construction of plasmid pPC2: Plasmid pCI-neo (purchased from Promega, USA) was digested with XhoI / SmaI, a 5.4kb fragment was recovered, and the oligonucleotide fragment L86 (5'-TCGAGATATCGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGACCC-3') was digested with T4DNA Ligase ligation, the ligation solution was transformed into E. coli DH5α, and cultured on LB solid medium containing anka penicillin (Ap, 100ug / ml) for 18-24 hours, picked 1 white transformant, and extracted the plasmid, which was pPC2.

[0026] The composition of the LB solid medium is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride, 1.5% agar, and 97.5% distilled water.

[0027] 2) Construction of plasmid pCFC: with Edwardsiella tarda TX1 as a template, PCR with the following primers: F8 (5'- AGTA CT GCCACCATGGCACAAGTAATCAACA...

Embodiment 2

[0064] 1) DNA vaccine preparation:

[0065] The plasmid pPC2 obtained in step 1 of Example 1 was transformed into Escherichia coli and cultivated in the LB liquid medium containing 100ug / ml Ap for 18-24 hours. Company”, Beijing) to extract the plasmid pPC2 for use;

[0066] Then the Edwardsiella tarda DNA vaccine shown in the nucleotide sequence in the sequence table 1 SEQ ID No.1 obtained in Example 1 is transformed into Escherichia coli and cultivated in the LB liquid medium containing 100ug / ml Ap for 18- Within 24 hours, use an endotoxin-free plasmid extraction kit (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) to extract the Edwardsiella tarda DNA vaccine shown in the base sequence in Sequence Table 1 SEQ ID No.1, and wait until use.

[0067] The above-mentioned obtained plasmid pPC2 and the Edwardsiella tarda DNA vaccine shown in the base sequence in Sequence Table 1 SEQ ID No.1 were dissolved in PBS respectively to a concentration of 200ug / ml, whi...

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PUM

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Abstract

The invention relates to the fields of molecular biology and immunology, more particularly to a DNA vaccine for Edwardsiella tarda and construction and an application thereof. The vaccine is shown as base sequence in a sequence table of SEQ ID No.1. The preparation method thereof comprises the steps of: constructing eukaryotic expression plasmids pPC2 and pCFC, and on this basis, constructing Edwardsiella tarda DNA vaccine plasmid pFXV6. The resultant DNA vaccine suspension has the immune protection effect on Edwardsiella tarda. The resultant DNA vaccine of the invention can reach high protection effect by one-time immunity. The immune protection effect of the DNA vaccine of the invention to Edwardsiella tarda runs up to 74%.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to an Edwardsiella tarda DNA vaccine and its construction and application. Background technique [0002] Edwardsiella tarda is a Gram-negative bacterium that infects humans, animals, and fish, including economically important farmed species of carp, tilapia, turbot, flounder, and eel. Edwardsiosis caused by Edwardsiella tarda causes huge economic losses to the aquaculture industry in the world (including my country) every year. Therefore, Edwardsiella tarda has been recognized as a serious aquaculture pathogen. [0003] At present, there is no commercial Edwardsiella tarda vaccine in the world. Vaccines against Edwardsiella tarda that are effective in the laboratory include inactivated whole-bacteria vaccines and recombinant subunit vaccines. The advantage of inactivated vaccines is safety, but its disadvantage is that the immune effect is poor. Recombinant subuni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/02A61P31/04C12N15/70C12R1/19
Inventor 孙黎焦绪栋
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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