Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis

doi:10.3969/j.issn.1673-8640.2014.06.007

Abstract

Abstract:

Objective To construct the prokaryotic expression vectors containing Mycobacterium tuberculosis(MTB) fusion gene. Meanwhile, the inducible expression of fusion protein recombinant dimer ESAT-6 (rdESAT-6) was performed in the expression systems of Mycobacterium smegmatis. The fusion protein was purified, and the antigenic specificities of fusion protein was analyzed by Western blot. The significance of fusion proteins for the diagnosis of tuberculosis was evaluated. Methods The 2×esat-6 gene was amplified with DNA vaccine HG856A₂. The 2×esat-6 gene was then cloned into plasmid pMF41 for the construction of prokaryotic expression vector pMF41-2×esat-6. The recombinant plasmid pMF41-2×esat-6 was electroporated into Mycobacterium smegmatis. The target gene was induced to express fusion protein rdESAT-6. The antigenic specificities of purified fusion protein was analyzed by Western blot with mouse antiserum against rdESAT-6. The sensitivity and specificity of these fusion proteins for the diagnosis of tuberculosis were assayed by enzyme-linked immunosorbent assay (ELISA) and tuberculosis dot immunogold filtration assay (TB-DOT) test kit for 126 tuberculosis patients and 42 healthy controls. Results The prokaryotic expression vector pMF41-2×esat-6 was constructed successfully, and the related fusion protein was expressed in the form of inclusion body. The fusion protein was purified, and the purity of these proteins were great than 95%. The fusion protein was antigenic with ESAT-6 mouse antiserum. The sensitivities of the 126 tuberculosis patients and negative cases of serological diagnosis were 79.75% (63/79) and 61.70% (29/47), respectively, which were better than those of TB-DOT test kit [62.03% (49/79) and 44.68% (21/47),P<0.05] . The specificity was both 95.24% (40/42). Conclusions The fusion proteins of MTB are successfully prepared in this study with good sensitivity and specificity, which can be used for the diagnosis of tuberculosis.

Key words: Mycobacterium tuberculosis, ESAT-6, Recombinant dimer ESAT-6, Fusion protein, Immunology diagnosis

CLC Number:

R446.5

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GUO Jian,FAN Qiwen,FAN Xiaoyong, MA Hui, QIAN Xueqin, HU Xiangnan, WU Wenjuan. Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis[J]. , 2014, 29(6): 607-612.

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