刚地弓形虫Peroxiredoxin基因的克隆及其融合蛋白的高效表达和纯化

Cloning,over-expression and purification of the recombinant peroxiredoxin protein of Toxoplasma gondii

目的克隆刚地弓形虫Prx基因,用IPTG诱导表达Prx融合蛋白并进行纯化。方法用PCR扩增目的基因片段并克隆至pGEX-6P-1载体,构建pGEX-6p-1/TgPrx原核表达载体;IPTG诱导表达Prx融合蛋白,表达产物进行SDS-PAGE和Western blot分析,GST亲和层析纯化融合蛋白。结果从弓形虫RH株DNA中扩增Prx基因,成功构建了弓形虫重组质粒pGEX-6p-1/TgPrx,并在大肠埃希菌(E.coli)中得到高效表达,表达的融合蛋白分子质量为51ku,该蛋白可被鼠抗弓形虫血清特异性识别。结论原核表达具有生物学活性的弓形虫重组Prx蛋白(rTgPrx),该蛋白有望用于弓形虫病免疫学检测。

Objective To clone the Peroxiredoxin（Prx） gene of Toxoplasma gondiiand to generate a prokaryotic expression vector to obtain a fusion protein of Prx with a GST tag Methods Prx gene fragments amplified by PCR were cloned into the pGEX-6P-1 vector to construct the prokaryotic expression vector pGEX-6p-1/TgPrx.Expression of the recombinant protein Prx（rTgPrx） was then induced by IPTG in E.coli BL21.Expressed products were purified by affinity chromatography and identified by SDS-PAGE,and Western blotting.Results The fusion protein rTgPrx with a molecular weight of 51 ku was efficiently expressed in E.coli BL21,and bound specifically to mouse polyclonal antibodies against Toxoplasma gondii.Conclusion A prokaryotic expression vector that expresses the recombinant protein Prx was successfully created and may provide the foundation for producing large quantities of rTgPrx to clinically diagnose human toxoplasmosis.