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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 purify Mycobacterium tuberculosis Ag85A protein by using Pichia pastoris in order to obtain specific antigens that efficiently stimulate γ-interferon production by T lymphocyte. Methods The Ag85a gene was amplified by PCR, and the recombinant plasmid pPic9k-85a was constructed. The recombinant plasmid pPic9k-85a was transformed into Pichia pastoris by electroporation. The target protein was purified by ammonium sulfate precipitation and ion exchange chromatography after geneticin G418 resistance screening. Finally, Ag85A expressed in E. coli and Pichia pastoris were respectively tested by Elispot as antigen stimulator for the ability of stimulating IFN-γ production by T lymphocytes of BCG-immunized mice. Results The Ag85A protein was successfully cloned and expressed in Pichia pastoris. The results of Elispot assay showed that Ag85A protein produced by Pichia pastoris was more effective than the Ag85A protein produced by E. coli in the stimulation of mouse T lymphocytes (P <0.05) More immune spots. Conclusion Ag85A protein expressed by Pichia pastoris can stimulate T lymphocytes more efficiently and can be used to detect the cellular immune response induced by Mycobacterium tuberculosis infection.