结核分枝杆菌Ag85A在毕赤酵母中的表达、纯化及酶联免疫斑点试验研究

The gene expression of Mycobacterium tuberculosis Ag85A in Pichia pastoris and enzyme-linked immunospot assay

目的利用毕赤酵母表达和纯化结核分枝杆菌Ag85A蛋白,以期得到高效刺激T淋巴记忆细胞产生γ-干扰素的特异性抗原。方法用PCR扩增得到Ag85a基因,构建出重组质粒pPic9k-85a,通过电转化进入毕赤酵母,经遗传霉素G418抗性筛选、硫酸铵沉淀浓缩及离子交换层析纯化得到目的蛋白。最后以大肠杆菌和毕赤酵母表达的Ag85A分别为抗原刺激物进行酶联免疫斑点(Elispot)试验,分析其刺激BCG免疫小鼠T淋巴细胞产生γ-干扰素(IFN-γ)的能力。结果在毕赤酵母中成功克隆并稳定表达了Ag85A蛋白,Elispot检测结果表明,毕赤酵母比大肠杆菌产生的Ag85A蛋白,对小鼠T淋巴细胞的刺激效果更加明显(P<0.05),能产生更多的免疫斑点。结论毕赤酵母表达的Ag85A蛋白可以更高效的刺激T淋巴细胞,可用于检测结核杆菌感染诱导的细胞免疫应答。

Objective To express and purity Mycobacterium tuberculosis Ag85A in Pichia pastoris and the application of purified antigen for the detection of cellular immunity. Methods The Mycobacterium tuberculo- sis Ag85a gene was amplified by polymerase chain reaction and then cloned into the vector pPic9k. Recombi- nant plasmid was transformed into Pichia pastoris by electroporation. High production bacterial strain was screened by G418. After fermentation, the broth was precipitated by ammonium sulfate and Ag85A was purified by ion exchange column. The spleen lymphocytes from the mice immunized by BCG were stimulated with Ag85A expressed in Pichia pastoris and E. coli for Elispot （enzyme-linked immunospot assay）. The different ability on stimulating cells for the production of IFN-Υ was analyzed. Results The Mycobacterium tuberculosis Ag85A was successfully expressed in Pichia pastoris, and can intensively stimulate T cells from the mice im- munized by BCG to produce more spots. Conclusion The Mycobacterium tuberculosis Ag85A antigen ex- pressed in Pichia pastoris can intensively stimulate T cells to product more IFN-7 in Elispot for the detection of cellular immunity in Mycobacterium tuberculosis infection.