弓形虫肌动蛋白表达及其抗血清制备

Expression of and preparation of polyclonal antibodies against actin of Toxoplasma gondii

目的体外制备弓形虫肌动蛋白(actin)及其多克隆抗体。方法以弓形虫cDNA链为模板,PCR扩增actin基因,亚克隆至pET30a载体,转化到大肠埃希菌BL21中;异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达actin-His6蛋白;用纯化的蛋白加免疫佐剂免疫SD大鼠,收集抗血清,制备多克隆抗体,抗体亲和纯化柱纯化并分析抗体的效价。结果 actin基因的PCR扩增产物经1.0%琼脂糖凝胶电泳,结果显示,在1 000 bp附近有一条带,与目的基因(actin cDNA长度为1 131 bp)大小相符;actin/pET30a重组质粒经BamHI和XhoI双酶切,1.0%琼脂糖凝胶电泳分析酶切产物,在1 000和5 000 bp附近可见条带;actin-His6蛋白在3～4 h时表达量达到峰值;蛋白可溶性分析显示,actin-His6蛋白主要以包涵体的形式存在;获得了抗actin-His6蛋白的大鼠源性抗血清。结论弓形虫actin蛋白在原核系统中得到了表达,用该蛋白免疫动物后获得了抗血清。

Objective To prepare the actin of Toxoplasma gondii （T. gondii） and the polyclonal anttbocly agamst the protein. Methods Actin gene was obtained from cDNA library of T. gondii by PCR amplification and subcloned to vector pET30a. The fusion protein actin-His6 was expressed in E. coli upon isopropyl-13-D-thiogalactoside（IPTG） induc- tion and then purified with affinity chromatography. The Sprague-Dawley （SD） rats were immunized with actin-His6 to produce polyclonal antibodies. Results The actin gene was obtained and the recombinant plasmid actin/pET30a was constructed successfully and expressed as a fusion protein. Serum were prepared from rats immunized with actin-His6 and purified with affinity chromatography to produce polyclonal antibodies with a antibody titer of 1：4000. Conclusion The protein actin-His6 was expressed in vitro. Serum and the polyclonal antibody against the protein were prepared, which may provide a foundation for further studies on the invasion mechanism of T. gondii.