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目的构建弓形虫P30基因表达载体并获得重组表达蛋白。方法将弓形虫P30基因的开读框用PCR扩增,NcoⅠ和HindⅢ酶切后,与同样酶切的表达质粒pET30a(+)经T4连接酶连接,然后转化到DH5α中。菌液经PCR扩增和质粒酶切及基因测序鉴定后,将阳性重组质粒转化到大肠埃希菌BL21(DE3)中,经IPTG诱导,表达产物用SDS PAGE和Westernblot进行鉴定。结果扩增的P30基因片段为750bp,重组质粒诱导表达产物分子质量单位为30ku,与理论值相符。Westernblot确认重组质粒表达蛋白与小鼠抗弓形虫单克隆抗体(P30McAb)发生特异性反应。结论成功构建重组体并获得弓形虫主要表面抗原P30的高效表达产物,为弓形虫病的诊断和疫苗研究奠定了基础。
Objective To construct the Toxoplasma gondii P30 gene expression vector and obtain the recombinant protein. Methods The open reading frame of P30 gene of Toxoplasma gondii was amplified by PCR, digested with Nco I and Hind Ⅲ, ligated with the same digested pET30a (+) T4 ligase and then transformed into DH5α. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) by PCR amplification, plasmid digestion and gene sequencing. The recombinant plasmid was induced by IPTG and identified by SDS PAGE and Western blotting. Results The amplified P30 gene fragment was 750bp, and the molecular mass unit of the recombinant plasmid induced expression was 30ku, which was consistent with the theoretical value. Western blot confirmed that the recombinant plasmid expression protein and mouse anti-Toxoplasma monoclonal antibody (P30McAb) specific reaction. Conclusion The successful construction of the recombinant plasmid and the high expression of the major surface antigen of Toxoplasma gondii P30 laid the foundation for the diagnosis and vaccine research of toxoplasmosis.