Mycobacterium tuberculosis H37Rv new gene as well as encoding protein and application thereof

A Mycobacterium tuberculosis, protein-encoded technology, applied in the field of antigen antibody detection, can solve the problems of time-consuming, low sensitivity, high false positive, etc.

Active Publication Date: 2021-08-20
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the diagnosis of tuberculosis mainly relies on 1) sputum bacteriological examination and chest X-ray imaging examination, but there are shortcomings of low sensitivity or time-consuming; 2) immunological diagnosis, but due to the production of BCG vaccine or other environmental mycobacteria Cross-immune reactions, resulting in high false positives, especially in BCG vaccination areas
So far, there have been many kits similar to T-SPOT.TB or kits relying on protein chip immunodiagnostic auxiliary technology at home and abroad, but all of them are difficult to identify the effective and specific neoantigens of Mycobacterium tuberculosis due to the lack of core problems. Solve the problem of "positive cannot be confirmed, negative is difficult to rule out"

Method used

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  • Mycobacterium tuberculosis H37Rv new gene as well as encoding protein and application thereof
  • Mycobacterium tuberculosis H37Rv new gene as well as encoding protein and application thereof
  • Mycobacterium tuberculosis H37Rv new gene as well as encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Discovery and verification of the new coding gene Rv3350 of Mycobacterium tuberculosis H37Rv

[0051] A high-coverage proteome study of the Mycobacterium tuberculosis H37Rv strain was performed using deep-coverage proteome technology. The pAnno software developed by the Institute of Computing Technology, Chinese Academy of Sciences was used to translate the whole genome (NC_000962.3) file of H37Rv published at NCBI according to the rule of translating one amino acid from three consecutive nucleotides on the positive strand and the complementary strand, using the terminator to For the translation mode of the terminator, 141,851 open reading frames (Opening reading frame, ORF) were translated using the three special initiation codons ATG, GTG and TTG of MTB. This database was used to search the H37Rv high-coverage proteome data. In order to reduce the false positive rate, strict quality control was carried out at the two levels of annotated peptides and new pe...

Embodiment 2

[0059] Example 2: Verification of the specificity of the Rv3350 new coding gene identified by proteomics

[0060] (1) Design primers:

[0061] Mycobacteria sequences (nearly 200 species, 8,046 strains) that have been sequenced from the NCBI database were downloaded, and the Mycobacteria database was constructed. After comparing the local homologous sequences of the target gene, primers were designed for regions with homologous but different sequences ( image 3 ).

[0062] PCR-specific primers were designed using Primer Premier 5 to compare the differential regions of the 600bp sequence (SEQ ID NO.4) before and after the target gene. The primer sequences are as follows:

[0063] F1: 5'-GTCTCCGAAATTGGGAATCGCCGCG-3' (SEQ ID NO.5);

[0064] R1: 5'-CACCTGCCGAGTCCGTGTTCGATCA-3' (SEQ ID NO. 6).

[0065] The positional relationship between the above primers and the Rv3350 (-|3760359-3761516|) gene is shown below, where the subscript wavy line corresponds to the position of the pr...

Embodiment 3

[0076] Example 3: Prokaryotic cloning, expression, purification and mass spectrometric identification of Rv3350 (-|3760359-3761516|) gene

[0077] 3.1 Cloning of Rv3350(-|3760359-3761516|) gene

[0078] (1) Design primers:

[0079] Based on the CDS sequence of the Rv3350 (-|3760359-3761516|) gene shown in SEQ ID NO.1, PCR primers were designed using Primer Premier 5. EcoR I and Hind III restriction endonuclease sites were added to the upstream and downstream primers, respectively, to facilitate subsequent cloning construction. The primer sequences are as follows:

[0080] F2: 5'-GGAATTCGTGGACGTCGAACAGCGA-3' (SEQ ID NO.7);

[0081] R2: 5'-CCAAGCTTTCACTGGTGGGGACACT-3' (SEQ ID NO. 8).

[0082] (2) The total DNA of the M.tuberculosis H37Rv strain was provided by the Tuberculosis Division of the Institute of Infectious Diseases, Chinese Center for Disease Control and Prevention.

[0083] (3) Amplify the DNA fragment, carry out polymerase chain reaction (PCR), and use the above...

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Abstract

The invention relates to a novel coding gene Rv3350 (-|3760359-3761516|) of mycobacterium tuberculosis H37Rv and a coding protein of the novel coding gene Rv3350(-|3760359-3761516|). Part or all of protein encoded by Rv3350 has B cell antigenicity, and can be independently or jointly used for clinical rapid screening of pulmonary tuberculosis patients and prevent human bodies from being infected with the mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to the field of antigen and antibody detection, in particular to mycobacterium tuberculosis gene and its application. Background technique [0002] Tuberculosis is a major global public health problem, and Mycobacterium tuberculosis (MTB) is the pathogenic bacterium that causes human tuberculosis. According to the latest 2019 global tuberculosis report released by WHO, there were about 10 million new cases of active tuberculosis in 2018, and tuberculosis is still the number one killer of infectious diseases. In addition to active tuberculosis patients, about 1 / 4 of the world's population is infected with MTB and faces the risk of further developing active tuberculosis. Therefore, the establishment of rapid, accurate, specific, sensitive and cheap methods for tuberculosis detection and identification of Mycobacterium tuberculosis is a necessary prerequisite for effective treatment and control of the spread of tuberculosis. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/35C12Q1/689C12Q1/04G01N33/569A61K39/04A61P31/06
CPCC07K14/35C12Q1/689G01N33/5695A61K39/04A61P31/06G01N2333/35
Inventor 徐平戴二黑张瑶王红武舒佳常蕾高慧英
Owner ACADEMY OF MILITARY MEDICAL SCI
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