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目的原核表达弓形虫棒状体蛋白ROP38,并对重组蛋白rROP38的反应原性进行鉴定。方法根据已发表的ROP38基因序列设计引物,通过RT-PCR方法扩增弓形虫RH株的ROP38基因。将ROP38部分基因克隆到原核表达载体p ET-28a(+)后,重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达并优化表达条件,SDS-PAGE分析表达情况,并通过Western blot鉴定其反应原性。结果 SDS-PAGE显示在上清和包涵体均有rROP38表达,分子量约为43k D。Western blot结果显示,rROP38蛋白能被His标签抗体和感染弓形虫的人阳性血清识别,说明ROP38具有良好的反应原性,可以作为潜在的血清学诊断抗原。结论本研究成功获得了具有良好反应原性的弓形虫重组蛋白rROP38。
Objective To express Toxoplasma gondii ROP38 in prokaryotic cells and identify the recombinant protein rROP38. Methods According to the published sequence of ROP38 gene, primers were designed and the ROP38 gene of Toxoplasma gondii RH strain was amplified by RT-PCR. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, and induced by IPTG. The expression conditions were optimized and analyzed by SDS-PAGE. The expression of ROP38 was detected by Western blot Identification of its reaction. Results SDS-PAGE showed rROP38 expression in the supernatant and inclusion bodies, the molecular weight of about 43kD. Western blot results showed that rROP38 protein could be recognized by His-tag antibody and human positive serum of Toxoplasma gondii infection, indicating that ROP38 has good reactivity and could be used as a potential serological diagnostic antigen. Conclusion Toxoplasma recombinant protein rROP38 has been successfully obtained in this study.