Preparation method of Mycobacterium tuberculosis Ag85A oral DNA vaccine

A technology of mycobacterium tuberculosis and DNA vaccines, applied in the direction of recombinant DNA technology, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as poor stability, weak immune response, and large antigenic mass

Inactive Publication Date: 2012-11-21
SHENYANG UNIV
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Problems solved by technology

In the past, the simple application of naked DNA vaccines has great disadvantages, such as poor stability in the gastrointestinal tract after oral intake, and the immune response of the gastrointestinal mucosa to antigen presentation is weak, so the amount of antigen required for oral immunization is large, which limits application of oral vaccines

Method used

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Embodiment

[0041] A preparation method of Mycobacterium tuberculosis Ag85A oral DNA vaccine comprises three continuous steps, the specific method is as follows:

[0042] 1. Construction of recombinant eukaryotic expression plasmid pCDNA3.1+ / Ag85A

[0043] 1. Primer design

[0044] According to the Ag85A gene sequence in the gene library, the primers used for polymerase chain reaction: the upstream primer 5'-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3', containing the BamHI restriction site; the downstream primer 5'-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3' containing the XhoI restriction site, and After the open reading frame stop codon. Ag85A contains a signal peptide sequence. Make sure that the open reading frame of the cloned gene is correct.

[0045] 2. Mycobacterium tuberculosis H37Rv strain was inoculated with modified Roche medium, cultured at 37°C for 8 weeks, and a small amount of DNA was extracted as a template.

[0046] 3. PCR Amplification of Ag85A Gene

[0047] Pre-denaturatio...

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Abstract

Relating to a vaccine preparation method, the invention provides a preparation method of a Mycobacterium tuberculosis Ag85A oral DNA vaccine. The method comprises: designing a pair of PCR primers according to a gene sequence of Mycobacterium tuberculosis Ag85A, taking the DNA of a human Mycobacterium tuberculosis H37Rv standard virulent strain as a template, conducting PCR amplification to obtain an Ag85A gene, subjecting a recycled PCR product to enzyme digestion by restriction endonuclease Xhol and BamHI, then connecting the product with a eukaryotic vector pCDNA3.1+ through a T4DNA ligase action, proving an obtained positive clone as the Ag85A gene by a DNA sequencing identification, cloning the Ag85A gene to the downstream of a CMV promoter in the vector pCDNA3.1+, constructing a recombinant plasmid pCDNA3.1+ / Ag85A, which is then transformed into Escherichia coli and amplified, thus obtaining the Mycobacterium tuberculosis Ag85A oral DNA vaccine. The method lays a foundation for clinical application research of oral DNA vaccines.

Description

technical field [0001] The invention relates to a preparation method of a vaccine, in particular to a preparation method of a Mycobacterium tuberculosis Ag85A oral DNA vaccine. Background technique [0002] In recent years, with the increase of population flow, the spread of AIDS, and the emergence of new tuberculosis strains and multi-drug resistant strains, tuberculosis has changed from the previous trend of decreasing year by year, and has resurged. The number of cases has increased significantly, and the incidence rate has increased at a rate of 20% per year. Infectious disease with the highest mortality rate. Therefore, vaccination plays a key role in the control and elimination of tuberculosis. [0003] Currently, Bacillus Calmette-Guerin (BCG) is the only clinically used vaccine for preventing and controlling tuberculosis, and the preventive effect is not stable. BCG is a Mycobacterium bovis isolated from a cow suffering from tuberculous mastitis. After 230 consecut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/04A61K9/127A61P31/06C12N15/31C12N15/79
Inventor 姜燕
Owner SHENYANG UNIV
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