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Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof

A technology of ESAT-6 and Mycobacterium tuberculosis, which is applied in the field of bioengineering and diagnostic medicine, can solve the problems of lack of specific antigen, inability to clearly distinguish BCG immunity, environmental mycobacterium infection and pathogenic Mycobacterium tuberculosis infection, application limitations, etc. To achieve the effect of simple and easy method

Inactive Publication Date: 2010-08-18
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunological diagnosis is simple, fast and sensitive, and has good development prospects, but its application in the early diagnosis of tuberculosis is limited due to the lack of specific antigens
At present, the antigen contained in the tuberculin purified protein derivative (PPD) used for the diagnosis of tuberculosis is shared by pathogenic mycobacteria, mycobacteria in the environment and BCG (BCG vaccine strain), so PPD cannot clearly distinguish BCG immune , environmental mycobacterial infection and pathogenic Mycobacterium tuberculosis infection, the differential diagnosis value is not high

Method used

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  • Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof
  • Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof
  • Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0041] Construction of Mycobacterium tuberculosis ESAT-6 Recombinant Dimer (rdESAT-6) Prokaryotic Expression Vector pET2E6

[0042] Using the DNA vaccine HG856A as a template, the 2×esat-6 gene was amplified by PCR, digested with BamHI-HindIII double enzymes and cloned into the pET28a plasmid to obtain the prokaryotic expression plasmid pET2E6( figure 1 ). The pET2E6 recombinant plasmid was digested with BamHI-HindIII double enzyme digestion, and the correct clone was sequenced ( figure 2 ).

Embodiment 2

[0044] Expression, Purification and Specific Identification of Mycobacterium tuberculosis ESAT-6 Recombinant Dimer (rdESAT-6)

[0045] The recombinant expression plasmid pET2E6 was transformed into Escherichia coli BL21(DE3), inoculated in LB medium containing 50 μg / mL of kanamycin, cultured overnight at 37°C, and transferred to 2% kanamycin-containing medium the next day. In LB medium, shake culture until A600 is about 0.4, add IPTG to a final concentration of 1 mmol / L, induce at 37°C for 4 hours, collect the bacteria by centrifugation, and analyze the expression of the target protein by 15% SDS-PAGE. After a large amount of induction, the collected bacterial pellet was resuspended in the lysate, and ultrasonically disrupted in an ice bath; the inclusion body precipitate was collected by centrifugation; the precipitate was washed twice with the inclusion body washing solution; the washed inclusion body was washed with the inclusion body dissolving solution Fully dissolve, cen...

Embodiment 3

[0046] Example 3. Serological diagnostic application of ESAT-6 recombinant dimer (rdESAT-6)

[0047] With the average OD value +2S of 24 normal human sera as the normal limit value and rdESAT-6 purified protein as the antigen, routine ELISA detection was performed on 20 tuberculosis patient sera and 24 normal human sera. 0.25 μg / mL rdESAT-6 purified protein was used to coat 100 μL / well of a 96-well microtiter plate (Greiner Bio-one), and the sera of normal people and tuberculosis patients were mixed with 1×PBS-T buffer containing 0.5% BSA 1:1000 After dilution, HRP-labeled goat anti-human IgG (1:12500) was used as the secondary antibody, and the anti-tuberculosis antibody level contained in the serum was determined by conventional ELISA method. The results of ELISA showed that 6 of 20 tuberculosis patient sera were detected positive, with a sensitivity of 30%; 1 positive was detected in 24 normal human sera, with a specificity of 95.8% (Fig. 5).

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Abstract

The invention belongs to the field of biological engineering and diagnostic medicine, in particular to a mycobacterium tuberculosis ESAT-6 recombinant dipolymer, a preparation method thereof, an antigenicity analysis and an application thereof in the serodiagnosis of tuberculosis. The invention uses the DNA of a chimeric gene nucleic acid vaccine HG856A plasmid containing 2 * esat-6 copies as the template, obtains 2 * esat-6 gene segments by PCR amplification, and clones, expresses and purifies an ESAT-6 recombinant dipolymer protein in E.coli. The protein is connected with the two ESAT-6 monomers through amino acid Tyr and Val which are coded by the AccI restriction enzyme cutting site, i.e. GTC TAC, carries six* His tags at the N end, and can be purified by nickel-chelate affinity chromatography. Experiments confirm that the purified recombinant dipolymer has favorable reactogenicity and antigenic specificity. The recombinant dipolymer can be used as an antigen for the early diagnosis of the tuberculosis, including the serodiagnosis of ELISA, ELISPOT or the like, or can be used as an antigen for a skin test.

Description

technical field [0001] The invention belongs to the fields of bioengineering and diagnostic medicine. In particular, it relates to a recombinant dimer of Mycobacterium tuberculosis ESAT-6 (rdESAT-6) and its preparation method, antigenicity analysis and application in serological diagnosis of tuberculosis. Background technique [0002] Tuberculosis is a major public infectious disease of worldwide concern. As early as 1993, the World Health Organization (WHO) declared a state of emergency for the tuberculosis epidemic because of the increasing number of tuberculosis cases worldwide. According to statistics, at present, about 1 / 3 of the world's population (1.86 billion) carries Mycobacterium tuberculosis, with about 8 million new cases and 2 million deaths from tuberculosis every year; there are about 6 million active tuberculosis patients in my country, and there are 250,000 people died from tuberculosis. According to the 2005 infectious disease epidemic situation announced...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/70C12N15/74G01N33/53C12R1/32C12R1/19
Inventor 范小勇郭建卢水华吴文娟
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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