Marker for tuberculosis diagnosis and application of marker

A technology for markers and tuberculosis, applied in the field of tuberculosis, can solve problems such as the difficulty of obtaining highly sensitive detection markers, and achieve high specificity and high affinity effects

Active Publication Date: 2019-11-12
SHANGHAI PULMONARY HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IHC detects protein, which has better sensitivity than the detection of bacteria itself, and is more convenient to observe. However, how to improve the intensity and range of IHC specific chromogenic signal, and improve the detection sensitivity and specificity is an urgent need to solve. The main difficulty is that it is difficult to obtain highly sensitive and specific detection markers

Method used

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  • Marker for tuberculosis diagnosis and application of marker
  • Marker for tuberculosis diagnosis and application of marker
  • Marker for tuberculosis diagnosis and application of marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This example is the identification and relative quantitative analysis of the supernatant protein components of Mycobacterium tuberculosis cultured in vitro under aerobic and anaerobic culture conditions, and further verification of the transcriptional expression level detection of Rv0859 under anaerobic culture conditions, which includes the following The above steps:

[0048] 1. Preparation of supernatant protein cultured by Mycobacterium tuberculosis under aerobic and anaerobic culture conditions in vitro:

[0049] The internationally recognized Wayne model was used to establish an in vitro anaerobic culture model of Mycobacterium tuberculosis, see literature for details. The details are as follows: get 2 conical screw cap flasks with side arms produced by Wheaton Company, add 200ml culture solution (Mie's 7H9 liquid medium+10% nutrient additive+0.4% Tween 80) into the aerobic culture bottle, anaerobic culture Add 400ml culture medium to the bottle. Mycobacterium tu...

Embodiment 2

[0062] This embodiment is the preparation of Mycobacterium tuberculosis antigenic protein Rv0859 and anti-Rv0859 polyclonal antibody, which comprises the following steps:

[0063] 1. Prokaryotic expression of Rv0859 in Escherichia coli:

[0064] First, using the extracted H37Rv genomic DNA, the gene coding fragment of protein Rv0859 was obtained by PCR cloning, and the fragment was connected to the pET-28a-SUMO vector (purchased from Invitrogen) by Invitrogen's ligase, and transformed into E. coli DH5-Alpha Amplify, extract the plasmid and identify it correctly by sequencing. Transform the vector into Escherichia coli Rosetta for amplification. When the OD600 is 0.5-0.6, add 0.8mM IPTG to induce at 37°C for 4 hours, collect the bacteria by centrifugation at 4000rpm, and identify the expression by protein electrophoresis. Depend on Figure 4 The results demonstrated that the prepared Rv0859 protein was of the correct size. Figure 5 In order to express the identification resu...

Embodiment 3

[0076] This embodiment uses the anti-Rv0859 polyclonal antibody prepared in Example 2 to detect the secretion level of Rv0859 protein in anaerobic culture conditions, which includes the following steps:

[0077] Same as Example 1, collect H37Rv under anaerobic / aerobic conditions and filter supernatant protein, use the anti-Rv0859 polyclonal antibody prepared in Example 2, Western Blot detection found that the level of Rv0859 protein was significantly up-regulated under anaerobic conditions (See Figure 9 ), verified the results of proteomics detection, and proved that the secretion level of Rv0859 was significantly increased under anaerobic conditions.

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Abstract

The invention relates to mycobacterium tuberculosis Rv0859 protein as a diagnosis marker in tuberculosis diagnosis. The Rv0859 protein is an amino acid sequence shown in SEQ ID No.1 or a variant of the sequence, which is protein having the same function. The invention also relates to preparation of an antibody for resisting Rv0859 as well as detection application of the anti-Rv0859 antibody as anactive component in tuberculosis diagnosis. A quantitative proteomics method finds that secretion level of the Rv0859 protein is significantly improved under an anaerobic culture condition and the Rv0859 can be used as the marker for diagnosis of tuberculosis; recombination expression in vitro is performed on the Rv0859, recombinant protein is used as an antigen to immunize animals, high-concentration pure anti-Rv0859 antibody is obtained, and the antibody can specifically detect Rv0859 protein of mycobacterium tuberculosis; and on the basis that the effective antibody is obtained, an immunohistochemical detection method with Rv0859 as the detection marker is established, which indicates that a new immunohistochemical detection method for tuberculosis can be established by the anti-Rv0859antibody.

Description

technical field [0001] The invention relates to the technical field of tuberculosis, in particular to a marker for diagnosis of tuberculosis and its application. The marker is Rv0859 protein. Background technique [0002] Tuberculosis is an infectious disease controlled by WHO, and it is still a public health problem that seriously endangers people's health. According to relevant statistics, about 1 / 3 of the world's population is infected with Mycobacterium tuberculosis. There are about 20 million tuberculosis patients, 8 million to 10 million new cases each year, and about 2 million people die of tuberculosis every year. The sum of deaths from infectious diseases. The tuberculosis epidemic in my country is extremely severe. There are currently 500 million people infected with tuberculosis, accounting for 1 / 4 of the world's total. Every year, 130,000 people die from tuberculosis, which is twice the total number of deaths from other infectious and parasitic diseases. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C07K16/12C07K16/06C07K1/22G01N33/569
CPCC07K14/35C07K16/065C07K16/1289G01N33/5695
Inventor 戈宝学杨华刘忠华黄晓辰王洁
Owner SHANGHAI PULMONARY HOSPITAL
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