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目的原核表达刚地弓形虫过氧化物氧化还原酶(peroxiredoxin,Prx)并制备多克隆抗体。方法 PCR技术扩增弓形虫cDNA中的prx基因,克隆至pET-28a(+)载体,构建prx/pET-28a(+)重组表达载体,转化至大肠埃希菌(E.coli)Rosetta中诱导表达。亲和层析纯化重组Prx蛋白,并制备兔多克隆抗体,蛋白印迹技术对多克隆抗体进行鉴定。结果成功从弓形虫cDNA中扩增出prx目的基因,构建了prx/pET-28a(+)重组质粒,获得抗Prx重组蛋白的多克隆抗体。蛋白印迹技术检测出弓形虫Prx的特异性条带。结论重组弓形虫Prx制备的多克隆抗体能检测Prx在弓形虫速殖子表达。
Objective To prokaryotic express Toxoplasma gondii peroxidase (peroxiredoxin, Prx) and prepare polyclonal antibodies. Methods The prx gene of Toxoplasma gondii cDNA was amplified by PCR and cloned into pET-28a (+) vector. The prx / pET-28a (+) recombinant vector was constructed and transformed into Escherichia coli Rosetta expression. Recombinant Prx protein was purified by affinity chromatography and polyclonal antibody was prepared. The polyclonal antibody was identified by Western blotting. Results The target gene prx was successfully amplified from Toxoplasma gondii cDNA. The recombinant plasmid prx / pET-28a (+) was constructed and the polyclonal antibody against Prx protein was obtained. Western blot detection of Toxoplasma gondii Prx specific bands. Conclusion The polyclonal antibody against Prx of Toxoplasma gondii can detect the expression of Prx in Toxoplasma gondii tachyzoites.