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Mycobacterium M. tuberculosis holoprotein chip and application thereof

A technology of mycobacterium tuberculosis and combined mycobacteria, applied in the biological field, can solve the problems of high GC content in the genome, difficult clinical prevention, slow growth, etc., and achieve the effect of improving research efficiency, simplifying research design and operation process

Active Publication Date: 2014-06-25
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The tuberculosis epidemic is serious, but there are difficulties in clinical prevention and control: 1) The detection rate of tuberculosis is low due to the absence of specific tuberculosis biomarkers and sensitive and rapid detection methods; 2) There is currently no effective vaccine for tuberculosis prevention
Although the popularization of BCG has greatly reduced the morbidity and mortality of tuberculosis in newborns and adolescents, it has problems such as short protection time, great differences among different populations, and ineffectiveness for adults.
3) In the past 60 years, no new anti-tuberculosis drugs have caused problems such as drug resistance, adverse reactions, and insufficient efficacy
However, due to some particularities of tuberculosis research, such as extremely slow growth, difficult to break, high GC content in the genome, difficult protein recombination and expression, and pathogenic bacteria must be operated in P3 laboratories, basic research on tuberculosis and mycobacterium tuberculosis long lag

Method used

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  • Mycobacterium M. tuberculosis holoprotein chip and application thereof
  • Mycobacterium M. tuberculosis holoprotein chip and application thereof
  • Mycobacterium M. tuberculosis holoprotein chip and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of whole protein chip

[0036] (1) High-throughput preparation of Mycobacterium tuberculosis proteins

[0037] 1. Express

[0038] The genetically engineered Saccharomyces cerevisiae uses galactose to induce overexpression, and the specific process is as follows:

[0039] First from the Mycobacterium M.tuberculosis H37Rv strain (Beijing strain) (Nature1998Nov12;396(6707):190; the public can obtain from Institute of Biophysics, Shanghai Jiao Tong University, Chinese Academy of Sciences Wuhan Virus Research Institute, Guangdong Tibikang Biotechnology Co., Ltd.) and CDC1551 strain (J Bacteriol.2002October;184(19):5479–5490; the public can obtain from Guangdong Tibikang Biotechnology Co., Ltd..), respectively 3749 proteins of H37Rv strain and 419 protein gene coding fragments of CDC1551 strain were obtained by PCR cloning, a total of 4168 proteins, and the gene coding fragments were connected to the pDONR221 vector (purchased from Invitrogen)...

Embodiment 2

[0200] Embodiment 2, application whole protein chip detects serum

[0201] (1) Preparation of serum samples to be tested

[0202] The serum of tuberculosis patients (infected with Mycobacterium tuberculosis) and the serum of healthy people (Guangdong Provincial Tuberculosis Control Center) were left at room temperature for 2 hours or overnight at 4°C and then centrifuged at 1000g for about 20 minutes. The supernatant can be tested immediately; or aliquot , and store the specimens at -20°C or -80°C, but repeated freezing and thawing should be avoided. Samples thawed at 4°C should be centrifuged again before testing.

[0203] (2) Preparation of various buffers and reagents involved in the diagnostic process

[0204] (1) Sample diluent (see Table 9): pH7.4 PBS solution

[0205] Table 9

[0206]

[0207] (2) Washing solution (see Table 10): PBST solution with pH 7.4

[0208] Table 10

[0209]

[0210] (3) Chip blocking solution (see Table 11): pH 7.4 PBS solution conta...

Embodiment 3

[0228] Example 3, the whole protein chip interacts with the small molecule compound c-di-GMP

[0229] c-di-GMP (cyclic diguanylic acid) is a ubiquitous second messenger molecule in bacteria that regulates diverse cellular events such as biofilm formation, virulence, motility and cell differentiation. Recent studies have found that Mycobacterium Tuberculosis and Mycobacterium smegmatis have enzymes that synthesize c-di-GMP and regulate c-di-GMP changes, but how c-di-GMP is regulated in Mycobacterium Tuberculosis has not been clear. Using the TB whole protein chip, c-di-GMP interacting proteins can be found globally, and their functions can be studied, so as to understand the role of c-di-GMP in Mycobacterium Tuberculosis more clearly and accurately.

[0230] 1. Closed chip

[0231] Prepare 20 mL of 3% BSA blocking solution with 1 × TBST, pour it into the sealing box, take two whole protein chips prepared in Example 1 from the -80°C refrigerator, put it in it, and put the seali...

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PUM

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Abstract

The invention discloses a mycobacterium M. tuberculosis holoprotein chip and an application thereof. The protein chip has at least one of the following functions (1) to (7); the following 4,168 proteins are connected to the protein chip, and each protein independently forms a detection point. Experiments show that the mycobacterium M. tuberculosis holoprotein chip consists of a substrate and proteins containing more than 95 percent of mycobacterium M. tuberculosis genome codes. The holoprotein chip is obviously significant for looking for a serum biomarker, a substrate interacting with a micromolecular compound c-di-GMP, a kinase substrate interacting with protein kinase and a substrate interacting with macrophage. Compared with an existing research means, the holoprotein chip has global and high-throughput screening characteristics, research design and operation processes are simplified, and the research efficiency is remarkably improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mycobacterium tuberculosis whole protein chip and its application. Background technique [0002] Tuberculosis is caused by Mycobacterium tuberculosis, which has plagued humans for 7,000 years. The emergence of BCG and antibiotics brought tuberculosis under control for a time. However, problems such as drug resistance and co-infection with HIV make tuberculosis resurgence worldwide. In 1993, WHO unprecedentedly declared a "global tuberculosis emergency", and in 1998 reiterated that "it is urgent to stop tuberculosis". At present, about 2 billion of the 6 billion people in the world are infected with Mycobacterium tuberculosis. There are 20 million tuberculosis patients and 3 million deaths each year. Tuberculosis has become the number one killer of infectious diseases. my country is one of the 22 countries with severe tuberculosis epidemics in the world, with an annual incidence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/5695G01N33/6803
Inventor 张先恩陶生策毕利军邓教宇张治平江何伟周盈谷晶郭书娟张鸿泰王绪德邓瑞萍王宗秀刘钟慧顾佳李阳李文娟
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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