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目的克隆弓形虫ROP18基因并构建该基因的原核表达系统。方法根据GenBank发表的弓形虫ROP18序列设计合成1对特异性引物,PCR扩增ROP18抗原基因,并亚克隆到pET-28a原核表达载体,重组质粒转入大肠埃希菌BL21感受态细胞,用IPTG诱导表达,重组蛋白用SDS-PAGE及Western blot鉴定。重组蛋白经纯化后免疫小鼠,ELISA法检测血清抗弓形虫IgG,并通过攻虫试验观察重组蛋白的免疫保护作用。结果成功构建了ROP18基因原核表达质粒pET-28a-ROP18,表达重组蛋白的分子质量单位约为55ku,该蛋白可被鼠抗弓形虫多抗血清识别;经ELISA检测,重组蛋白免疫小鼠产生了较高水平的血清抗体;攻虫试验中,重组蛋白免疫小鼠生存时间较对照组小鼠显著延长(P<0.05)。结论成功构建了ROP18基因原核表达质粒,表达的重组蛋白具有一定的免疫保护作用。
Objective To clone Toxoplasma gondii ROP18 gene and construct its prokaryotic expression system. METHODS: One pair of specific primers was designed and synthesized according to the ROP18 sequence of Toxoplasma gondii published in GenBank. The ROP18 gene was amplified by PCR and subcloned into prokaryotic expression vector pET-28a. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells and treated with IPTG Induction of expression, recombinant protein by SDS-PAGE and Western blot identification. Recombinant proteins were purified and immunized mice, serum anti-Toxoplasma gondii IgG was detected by ELISA, and the protective effect of the recombinant protein was observed by the test of helminth insects. Results The prokaryotic expression plasmid pET-28a-ROP18 of ROP18 gene was constructed successfully. The molecular weight of recombinant protein was about 55ku. The recombinant protein could be recognized by anti-T.gondii polyclonal antiserum. After immunization with recombinant protein, Higher level of serum antibody. In the test of worm attack, the survival time of the recombinant protein immunized mice was significantly longer than that of the control mice (P <0.05). Conclusion The prokaryotic expression plasmid of ROP18 gene was successfully constructed and the expressed recombinant protein has certain immunoprotective effect.