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目的通过cDNA文库筛选出弓形虫关键粘附蛋白基因—棒状体ROP2基因,在大肠埃希菌BL21(DE3)中表达,以获得重组蛋白。方法根据弓形虫棒状体ROP2基因开放阅读框设计引物,以弓形虫cDNA为模板进行PCR,纯化扩增产物经双酶切,进行TA克隆,再克隆到pET-30a中。重组质粒pET-30a-rop2经PCR鉴定后,转化到大肠埃希菌BL21(DE3)中,经IPTG诱导表达,用SDS-PAGE电泳鉴定表达产物。结果获得的1223bp的基因片段经PCR和酶切鉴定后与预期大小一致;SDS-PAGE电泳分析显示诱导菌在52ku处出现一条明显的蛋白带,与预期分子量相符。结论成功构建PET30a-ROP2,并在大肠埃希菌BL21(DE3)中高效表达,为进一步研究弓形虫粘附关键蛋白基因与肠上皮细胞的关系奠定基础。
OBJECTIVE: To screen the cDNA of Toxoplasma gondii, the key adhesion protein gene of Rodent Toxoplasma gondii, and to express it in Escherichia coli BL21 (DE3) to obtain the recombinant protein. Methods According to the open reading frame of ROP2 gene of Toxoplasma gondii, primers were designed and the Toxoplasma gondii cDNA was used as a template for PCR. The amplified product was double digested and cloned into pET-30a. After identification by PCR, the recombinant plasmid pET-30a-rop2 was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE electrophoresis. Results The 1223bp gene fragment was identified by PCR and restriction enzyme digestion. The result was consistent with the expected size. SDS-PAGE analysis showed that the induced strain showed a distinct protein band at 52ku, which was consistent with the expected molecular weight. Conclusion The successful construction of PET30a-ROP2 and its high expression in Escherichia coli BL21 (DE3) lay the foundation for further study on the relationship between Toxoplasma adhesion protein and intestinal epithelial cells.