结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性

doi:10.11843/j.issn.0366-6964.2014.05.016

Abstract

Abstract:

The aim of this study was to express the fusion protein ESAT6-CFP10 of Mycobacterium tuberculosis and evaluate its cellular immunoproperties in mice model as well as the detection potential for bovine tuberculosis.The fragment of esat6-linker-lhp was amplified using PCR and then constructed into the pET30a(+)-esat6-linker-lhp recombinant plasmid.Fusion protein of ESAT6-CFP10 was purified with His affinity chromatography column from strain of BL21(DE3)-pET30a(+)-esat6-linker-lhp after induced with IPTG.The purified fusion protein was then used to evaluate its cellular immunoproperties in mice model as well as the potential usage in IFN-γ assay for the detection of bovine tuberculosis.Results of SDS-PAGE and Western blotting implied that the fusion protein was expressed successfully and having good immunological reactivity.FACS analysis showed that there was a significant up-regulation of molecules both CD80 and CD86 expression on dendritic cells as well as CD69 on splenic CD4⁺ and CD8⁺ T cells，meanwhile，more IFN-γ specific secreting cell spots rather than those of IL-4 were detected by ELISPOT assay in C57BL/6 mice which injected with the fusion protein.IFN-γ test results of 479 cow blood samples showed that the fusion protein had the coincidence rates of 70.0% (positive) and 95.2% (negative) compared to IFN-γ test kit using PPD as stimulator.These data indicated the fact that this fusion protein had the capability of induce Th1 immune response in mice and provided a basis for the diagnosis of bovine tuberculosis.

CLC Number:

S852.618

MENG Chuang， WAN Ting， XU Zheng-zhong， SHAN Fa， XIE Xiao-li， SHEN Jun-song， FAN Feng， CHEN Xiang， JIAO Xin-an. Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 Protein and Its Cellular Immunoproperties[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(5): 789-794.

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Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 Protein and Its Cellular Immunoproperties

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