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Applications of Mycobacterium tuberculosis protein

A technology of Mycobacterium tuberculosis and protein is applied in the application field of Mycobacterium tuberculosis protein, which can solve the problems of cross-contamination, good sensitivity, and difficulty in meeting high sensitivity and specificity, and achieves the effect of high reliability.

Active Publication Date: 2021-11-23
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This test has been used in the clinical diagnosis of TB, but its specificity is poor and it often cross-reacts with environmental mycobacteria and Bacillus Calmette-Guerin (BCG)
(4) MTB nucleic acid amplification detection: This method has good sensitivity, and even bacteria with low copy number in the specimen can be detected, but there will be deviations in clinical application, such as improper specimen handling, cross-contamination of target sequences or amplification products, etc. prone to false negatives
This method obtains recombinant antigens through genetic engineering, and then uses indirect ELISA to detect the reactivity of recombinant antigens with patient serum, but this method is difficult to meet the requirements of high sensitivity and specificity

Method used

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  • Applications of Mycobacterium tuberculosis protein
  • Applications of Mycobacterium tuberculosis protein
  • Applications of Mycobacterium tuberculosis protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: ELISA detection test for specific binding of representative phages P1 to P4 to TB positive serum

[0029] 100 μL of TB-positive serum (100 μg / mL) was coated on a microplate (while setting up a negative serum control and a BSA control), and incubated overnight at 4°C in a humid chamber. After discarding the coating solution, block with blocking solution (5% skimmed milk powder) for 2 h, wash with PBST 8 times, and add 150 μL of amplified representative phage (3×10 11 pfu, the T7 phage whose surface displays the protein encoding the sequence shown in SEQ ID NO:1-4), incubated at 37°C for 2h, washed 8 times with PBST, added 1:1000 diluted HRP-labeled anti-T7 phage antibody, and incubated at 37°C After 2 hours, wash with PBST 8 times, add A and B chromogenic solutions respectively, keep away from light at 37°C for 15 minutes, and measure the absorbance value at 450nm (A450) with a microplate reader after adding the stop solution.

[0030] Such as figure 1 Shown...

Embodiment 2

[0031] Example 2: Dot immunoblotting test for specific binding of representative phages P1 to P4 to TB positive sera

[0032] The PVDF membrane soaked in methanol until transparent was washed with TBST (0.5% Tween 20), and 1 μL of amplified representative phage (10 11 pfu, surface-displayed T7 phage encoding the sequence protein shown in SEQ ID NO: 1-4), after drying at room temperature, put it in a 4°C refrigerator to seal overnight, wash the membrane 3 times with TBST, and add the preliminary purified TB positive serum ( 1:50), incubated at 37°C for 2 h, washed the membrane 6 times with TBST, added HRP-labeled goat anti-human IgG antibody (1:4 000) and soaked the membrane at 37°C for 1 h, washed the membrane 6 times with TBST, and then emitted light by ECL method. Develop and fix. At the same time, set up negative control, blank control, wild-type phage control and positive control.

[0033] In order to further confirm that representative phages P1-P4 can specifically comb...

Embodiment 3

[0034] Embodiment 3: express and purify recombinant RK

[0035] A prokaryotic expression vector containing RK gene was constructed, and recombinant ribokinase was expressed and purified. The results showed that a PCR product with a size of 975 bp was successfully amplified, which had 100% homology with the DNA sequence of the RK gene. The prokaryotic expression vector pET-28a(+)-RK( image 3 a), expressed and purified the recombinant protein (36kDa) containing histidine tag ( image 3 b); if image 3 As shown in c, SDS-PAGE analysis shows that the purified recombinant protein appears as a single band. These results indicated that the recombinant ribokinase full-length protein was successfully expressed and purified. As identified by Western blot, the recombinant RK can specifically bind to the anti-histidine tag antibody and tuberculosis positive serum ( Figure 4 ).

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Abstract

The invention relates to the field of biotechnology, and discloses the application of mycobacterium tuberculosis protein. In the present invention, ribokinase, fatty acyl-AMP ligase, cystathionine β-lyase and hypothetical protein in Mycobacterium tuberculosis protein are used as diagnostic antigens, and the corresponding antibodies in the serum to be tested are detected by indirect ELISA to determine the Whether the patient is infected with Mycobacterium tuberculosis, and further judge whether to suffer from tuberculosis, has high reliability, specificity and sensitivity, and can be used in the field of serological diagnosis of tuberculosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of mycobacterium tuberculosis protein. Background technique [0002] Tuberculosis (TB) is a chronic infectious zoonotic disease caused by the infectious pathogen Mycobacterium tuberculosis (MTB), and it is one of the most important infectious diseases threatening human health. MTB is mainly divided into human, bovine, African and mouse types. More than 90% of human tuberculosis is caused by infection with human-type MTB, and a few are caused by bovine and African types. MTB can invade various organ systems of the whole body, and the most common diseased part is the lung, and other organs such as the liver, bone, and brain can also be affected. MTB infection is divided into two states: latent and active. Active pulmonary tuberculosis refers to those with positive sputum smears: that is, MTB is excreted from the body, MTB reproduces actively, and has strong virulence....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569
Inventor 曾燚华王丽罗丹
Owner NANHUA UNIV
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