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Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof

A technology of mycobacterium tuberculosis and rv1284, applied in the field of biomedical in vitro diagnostic reagents, can solve the problems of low detection rate, complicated operation and high cost

Inactive Publication Date: 2012-10-10
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are many diagnostic methods for MTB infection, but the most commonly used clinical method still relies on the traditional sputum smear bacteriological examination, but the detection rate is low; MTB culture can be used as the "gold standard" for the diagnosis of tuberculosis, but the culture time is too long. Long, generally 4-6 weeks, it is difficult to meet clinical needs; Bactec technology shortens the culture time, but the cost is high, and it is difficult to popularize in a short time; X-ray and CT examinations only provide imaging possibilities for diagnosis; polymerase chain reaction (PCR) technology and other genetic diagnosis methods are still complex in operation as clinical diagnosis, require high technical and professional quality of personnel, and still cannot be used for rapid diagnosis of bacillus-negative tuberculosis

Method used

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  • Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof
  • Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof
  • Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0058] 1. Construction of recombinant plasmid pET32a-Rv1284

[0059] (1) Target gene primer design

[0060] Rv1284-F (SEQ ID No: 3): TA GGATCC AGCGGTTTCAAGGGC;

[0061] Rv1284-R (SEQ ID No: 4): ATT CTCGAG GGGCGTGACCTCGTTG;

[0062] The enzyme cutting sites are BamH I and Xho I, respectively.

[0063] (2) PCR amplification, cloning and sequence determination of the target gene

[0064] Mycobacterium tuberculosis H 37 Rv genomic DNA was used as a template, Rv1284-F and Rv1284-R were used as primers, and the Rv1284 protein gene was directly amplified by PCR using Taq enzyme (Bao Bioengineering (Dalian) Co., Ltd.). PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; (94°C, 30s; 60°C, 30s; 72°C, 40s) 30 cycles; extension at 72°C for 5 minutes; storage at 4°C. After the reaction, the target fragment was separated by 1% agarose gel electrophoresis, and then recovered with a DNA recovery kit (Invitrogen). Digested with BamH I and Xho I, cloned into pET32a plasmid...

Embodiment 2

[0067] In Example 2, Mycobacterium tuberculosis ESAT-6 antigen and 16kDa antigen were used as controls to evaluate the effect of recombinant Rv1284 antigen as a detection reagent on IgG in healthy control serum and tuberculosis patient serum.

[0068] The IgG reaction against Rv1284 in different serum samples was detected by ELISA method. The specific steps are: 96-well plate with antigen Rv1284 (2.5 μg / ml), ESAT-6 (recombinantly expressed by our research group according to the conventional technology described below) (5.0 μg / ml) or 16kDa antigen (our research group according to the following The conventional technique described for recombinant expression) (20 μg / ml) was coated overnight, the coating solution was discarded, washed 5 times with PBST, and dried. Add 300 μl of PBST containing 1% BSA to each well and incubate at 37°C for 2h. Spin dry, add 100 μl serum to be tested (1:50) diluted with PBST containing 1% BSA to each well, and incubate at 37°C for 0.5h. Discard the...

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Abstract

The invention provides a recombinant protein of a mycobacterium tuberculosis specific marker Rv 1284. The recombinant protein comprises an amino acid sequence as shown in SEQ ID NO. 1. The invention also provides a coding nucleotide sequence of the recombinant protein of the mycobacterium tuberculosis Rv 1284, a preparation method of the recombinant protein, and an application of the recombinant protein. According to the invention, a mycobacterium tuberculosis secretory protein Rv 1284 is first reported on the basis of the research results of immunomics. The mycobacterium tuberculosis secretory protein Rv 1284 can be applied to tuberculosis diagnosis, and is the tuberculosis marker with high immunocompetence; and the mycobacterium tuberculosis secretory protein Rv 1284 can be used for the tuberculosis immunological diagnosis, and has relatively high sensibility, and is simpler, more convenient, faster, safer and more reliable.

Description

technical field [0001] The invention belongs to the field of biomedical in vitro diagnostic reagents, in particular to a Mycobacterium tuberculosis specific marker Rv1284 recombinant protein, its preparation method and its application in tuberculosis immunodiagnosis. Background technique [0002] The development of a simple, rapid, accurate and reliable diagnosis of Mycobacterium tuberculosis (MTB) infection is of great importance for the control of tuberculosis. At present, there are many diagnostic methods for MTB infection, but the most commonly used clinical method still relies on the traditional sputum smear bacteriological examination, but the detection rate is low; MTB culture can be used as the "gold standard" for the diagnosis of tuberculosis, but the culture time is too long. Long, generally 4-6 weeks, it is difficult to meet clinical needs; Bactec technology shortens the culture time, but the cost is high, and it is difficult to popularize in a short time; X-ray a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70G01N33/68C12Q1/68C12R1/32
Inventor 张舒林张湘燕孙战强赵俊伟郭晓奎王洪海
Owner SHANGHAI JIAO TONG UNIV
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