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Application of mycobacterium tuberculosis protein

A Mycobacterium tuberculosis protein technology, which is applied in the application field of Mycobacterium tuberculosis protein, can solve the problems of cross-contamination, good sensitivity, and clinical application deviation, and achieve high reliability results

Active Publication Date: 2019-05-17
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 antigen through genetic engineering, and then uses indirect ELISA to detect the reactivity of recombinant antigen and patient serum, but this method is difficult to meet the requirements of high sensitivity and specificity

Method used

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  • Application of mycobacterium tuberculosis protein
  • Application of mycobacterium tuberculosis protein
  • Application 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] Coat 100 μL of TB positive serum (100 μg / mL) on the microplate (set up negative serum control and BSA control at the same time), and incubate 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 for 15 minutes at 37°C, 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] Wash the transparent PVDF membrane soaked in methanol with TBST (0.5% Tween 20), and add 1 μL of amplified representative phage (10 μL) dropwise to the membrane. 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, negative control, blank control, wild-type phage control and positive control were set up.

[0033] In order to further confirm that representative phages ...

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 demonstrate the successful expression and purification of the recombinant ribokinase full-length protein. As identified by Western blot, the recombinant RK can specifically combine with 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 application of a mycobacterium tuberculosis protein. According to the application of the mycobacterium tuberculosis protein, ribose kinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in the mycobacterium tuberculosis protein are used as a diagnostic antigen, a corresponding antibody in a serum to be detected is detected through an indirect ELISA so as to judge whether a person to be detected is infected with mycobacterium tuberculosis or not, whether the a person to be detected is suffered tuberculosis or not is further judged, high reliability, specificity and sensitivity are achieved, and the application of a mycobacterium tuberculosis protein can be applied to the field of serological diagnosis of the 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 type, bovine type, African type and mouse type. More than 90% of human tuberculosis is caused by infection with human type MTB, and a few are caused by bovine type and African type. MTB can invade various organ systems throughout the body, and the most common diseased part is the lungs, but it can also affect other organs such as the liver, bone, and brain. 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 ha...

Claims

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Application Information

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