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Antigen Protein and Application of Mycobacterium Tuberculosis

A technology of mycobacterium tuberculosis and antigenic protein, which is applied in the field of molecular diagnosis of infectious diseases and genetic engineering, can solve the problems of high infection rate and high cost of tuberculosis, and achieve the effect of low operating cost and accurate detection

Inactive Publication Date: 2011-11-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these techniques require high costs and highly skilled operators, limiting their adoption and application in low-income countries where TB infection rates are often high

Method used

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  • Antigen Protein and Application of Mycobacterium Tuberculosis
  • Antigen Protein and Application of Mycobacterium Tuberculosis
  • Antigen Protein and Application of Mycobacterium Tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: PCR amplification of the Mycobacterium tuberculosis antigenic protein coding gene

[0030] The applicant previously screened three gene fragments from the genome sequence of Mycobacterium tuberculosis, which can strongly react with the serum of tuberculosis patients. Their nucleotide sequences are respectively shown in the sequence listing SEQ ID NO: 1, 3, 5, and their coded amino acid sequences are respectively shown in the sequence listing SEQ ID NO: 2, 4, 6. Among them, the nucleotide sequence as shown in the sequence table SEQ ID NO: 1 is screened from the Rv1987 gene (NCBI accession number NP_216503.1, GeneID: 885815, gene annotation is possible chitinase), and the sequence length is 0.429kb , screened from the Rv3807c gene (NCBI accession number NP_218324.1, GeneID: 886134, the gene is annotated as a possible conserved transmembrane protein) to obtain the nucleotide sequence shown in the sequence table SEQ ID NO: 2, the sequence length is 0.498 kb, scr...

Embodiment 2

[0052] Example 2: Construction of a histidine tag fusion expression vector for the antigen gene of Mycobacterium tuberculosis

[0053] The general LB medium used in this example and the additional antibiotic kanamycin (shown in the specific steps) formula refer to J. Sambrook, EF. Fritsch, T. Mani Artis, Huang Peitang, Translated by Wang Jiaxi, Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002.

[0054] (1) The Mycobacterium tuberculosis antigenic protein coding gene PCR product obtained by embodiment 1 and the pET28a expression vector (vector map sees image 3 ) were digested by corresponding restriction endonucleases (purchased from Bao Biological Engineering (Dalian) Co., Ltd.);

[0055] (2) Ligate the digested product obtained in step (1) and the carrier overnight at 16°C;

[0056] The system of the ligation reaction is shown in Table 3:

[0057] The system of table 3 ligation reaction

[0058]

[0059] (3) Transformation of ligation products: ...

Embodiment 3

[0063] Example 3: Mycobacterium tuberculosis antigenic protein induced expression in Escherichia coli

[0064] The general LB medium used in this example and the additional antibiotic kanamycin (shown in the specific steps) formula refer to J. Sambrook, EF. Fritsch, T. Mani Artis, Huang Peitang, Translated by Wang Jiaxi, Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002.

[0065] (1) Transform the recombinant plasmids obtained in Example 2 into BL21(DE3) strains (purchased from Shanghai Chaoyan Biotechnology Co., Ltd.): Take 100 μl BL21(DE3) competent cells and add them to 1.5ml EP tubes, add Take 1 μl each of the recombinant plasmids obtained in Example 3 and mix well. After placing on ice for 30 minutes, heat shock at 42°C for 90 seconds and ice bath for 1 minute. Add 400 μl of LB medium, shake and culture at 37° C. at 200 rpm for 45 minutes. The revived E. coli liquid was centrifuged at 4000 rpm for 3 minutes, 400 μl of supernatant was discarded, th...

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Abstract

The invention belongs to the technical field of gene engineering and is related to the technical field of molecular diagnosis of infectious diseases. The invention relates to preparation of three kinds of antigen proteins of Mycobacterium tuberculosis, and application of the three kinds of antigen proteins in preparation of a tuberculosis diagnosis assay kit. The antigen proteins of the Mycobacterium tuberculosis have nucleotide sequences shown as SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 in a sequence table, and amino acid sequences encoded by the antigen proteins are shown as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 in the sequence table. Biological experiments indicate that the three kinds of antigen proteins of Mycobacterium tuberculosis can be applied to the preparation of the tuberculosis diagnosis assay kit.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and also relates to the technical field of molecular diagnosis of infectious diseases, in particular to three kinds of Mycobacterium tuberculosis antigenic proteins and their application in the preparation of tuberculosis diagnosis and detection kits. Background technique [0002] The threat of animal bacterial infectious diseases to human health is becoming more and more serious, which has an impact and influence on national economy and social stability. Mycobacterium tuberculosis (Mycobacterium tuberculosis, the main pathogenic bacterium of tuberculosis) is a pathogen that seriously endangers human health. One-third of the world's population carries Mycobacterium tuberculosis in a latent state, and there are about two million people every year. Died of tuberculosis (Raviglione M C. The TB epidemic from 1992 to 2002. Tuberculosis (Edinb), 2003, 83: 4-14.). In recent years, Mycobacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/31G01N33/569
Inventor 何正国李雨庆
Owner HUAZHONG AGRI UNIV
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