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Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof

A technology for mycobacterium tuberculosis and latent infection, which is applied in the field of medical immunology diagnosis, can solve the problems of false positive, false negative, and low efficiency of TST diagnosis of latent infection, and achieve low cost, auxiliary diagnosis and differential diagnosis, high sensitivity and specific effect

Inactive Publication Date: 2015-07-29
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TST is cheap, and it is mostly used as the main detection method in China. However, TST is affected by BCG and the immune status of the subjects, and it is prone to false positive and false negative results. Very low efficiency in diagnosing latent infection
IGRA has difficulty distinguishing between active and latent TB infection

Method used

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  • Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof
  • Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof
  • Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Construction of engineered bacteria by genetic engineering technology, induced expression and identification of purified recombinant protein LTBIPr1

[0062] 1. Design and synthesize a pair of primers for amplifying the LTBIPr1 epitope according to the sequence 1 in the sequence listing

[0063] Upstream primer (5' end contains restriction endonuclease Nhe I)

[0064] 5'-CTAGGCTAGCCACCATGGCGGAGCCAGCGGCGTG-3'

[0065] Downstream primer (5' end contains restriction endonuclease EcoR I)

[0066] 5'-CCGGAATTCTCATGGCGAGGCTTCCGGG-3'

[0067] Amplified fragment: 1035bp

[0068] 2. PCR amplification of LTBIPr1 gene

[0069] The above-mentioned upstream and downstream primers were used to amplify the LTBIPr1 gene under the action of Taq DNA polymerase and Mycobacterium tuberculosis H37RvDNA as a template. After hot start at 94°C for 10 minutes, DNA polymerase was added for PCR. The reaction program was: 94°C for 1 min, 65°C for 1 min, 72°C for 1 min, and 30 cycles; final...

Embodiment 2

[0113] 1. Construction of engineering bacteria expressing LTBIPr2 protein by genetic engineering technology:

[0114] 1. Design and synthesize a pair of primers for amplifying the LTBIPr2 epitope according to the sequence 1 in the sequence listing

[0115] Upstream primer (5' end contains restriction endonuclease Nhe I)

[0116] 5'-CTAGGCTAGCCACCATGGCGCAAACCGGCAAGC-3'

[0117] Downstream primer (5' end contains restriction endonuclease EcoR I)

[0118] 5'-CCGGAATTCTCACATCTCCTGGTTCTCG-3'

[0119] Amplified fragment: 1143bp

[0120] 2. PCR amplification of LTBIPr2 gene

[0121] The above-mentioned upstream and downstream primers are used to amplify the LTBIPr2 gene under the action of Taq DNA polymerase and Mycobacterium tuberculosis H37RvDNA as a template. After hot start at 94°C for 10 minutes, DNA polymerase was added for PCR. The reaction program was: 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute, and 30 cycles; finally, 72°C for 7 minutes. The 1143bp amplifi...

Embodiment 3

[0165] 1. Construction of engineering bacteria expressing LTBIPr3 protein by genetic engineering technology

[0166] 1. Design and synthesize a pair of primers for amplifying the LTBIPr3 epitope according to the sequence 1 in the sequence listing

[0167] Upstream primer (5' end contains restriction endonuclease Nhe I)

[0168] 5'-CTAGGCTAGCCACCATGGCCACGCAACGACCGAG-3'

[0169] Downstream primer (5' end contains restriction endonuclease EcoR I)

[0170] 5'-CCGGAATTCTTAGACCGCAACGGCAATC-3'

[0171] Amplified fragment: 378bp

[0172] 2. PCR amplification of LTBIPr3 gene

[0173] The above-mentioned upstream and downstream primers are used to amplify the LTBIPr3 gene under the action of Taq DNA polymerase and Mycobacterium tuberculosis H37Rv DNA as a template. After hot start at 94°C for 10 minutes, DNA polymerase was added for PCR. The reaction program was: 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and 30 cycles; finally, 72°C for 7 min. The 378bp amplified DNA fragmen...

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Abstract

The present invention relates to Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof, wherein the three Mycobacterium tuberculosis latent infection related proteins are LTBIPr1, LTBIPr2 and LTBIPr3, and the nucleotide sequences of the protein epitope are represented by the sequences 1-3 in the sequence table. According to the present invention, the genetic engineering technology is adopted to clone, express and purify Mycobacterium tuberculosis to obtain the three protein antigens related with Mycobacterium tuberculosis latent infection; the protein antigen is adopted as the tuberculosis specific cell stimulating agent to stimulate the peripheral blood mononuclear T-lymphocyte of a subject so as to make the T-lymphocyte secrete specific IFN-gamma, and then the spot number of the T-lymphocyte secreting the IFN-gamma is detected through the ELISpot technology, wherein the whole process takes two days; and according to the generated blue-purple spot number, the result is read with the naked eye or instrument, such that the Mycobacterium tuberculosis latent infection related proteins can be used for detection of whether a subject is infected with tubercle bacillus, and active tuberculosis assisted diagnosis and differential diagnosis.

Description

technical field [0001] The invention relates to mycobacterium tuberculosis latent infection-related proteins and their preparation and application, in particular to three kinds of mycobacterium tuberculosis latent infection-related proteins LTBIPr1, LTBIPr2 and LTBIPr3 prepared by applying genetic engineering technology and their preparation methods, belonging to medical immunology diagnosis technology field. Background technique [0002] Tuberculosis is one of the major infectious diseases that endanger human health worldwide. The prevalence of AIDS, the immigration of tuberculosis infected persons, and the poverty of some people have led to a rising trend in the incidence of tuberculosis in the United States and other developed countries in Europe and the United States. To add insult to injury. At present, there are about 20 million tuberculosis patients in the world, 8 to 10 million new tuberculosis patients are added every year, and the annual death toll is about 2 mil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/00C07K14/35C12N15/54C12N15/52C12N15/31C12N15/70G01N33/569G01N33/68
CPCC12N9/1205C07K14/35C12N9/00C12Y207/01011G01N33/5695G01N33/68
Inventor 吴雪琼白雪娟梁艳阳幼荣张俊仙
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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