摘要
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 a recombinant plasmid containing P30 gene of Toxoplasma gondii and obtain recombinant protein Methods P30 gene fragment was amplified from genomic DNA of T. gondii RH strain by PCR. The purified PCR fragment was ligated into the sites of pET-30a(+). The recombinant vector was transformed into Escherichia coli DH5α. The positive clones were screened and identified by PCR from bacteria directly and digested via double enzymes cut. Sequencing of recombinant plasmid was performed. The recombinant plasmid was transformed into E. coli BL21(DE3), the expression of protein was induced by IPTG. The expression product was identified by SDS-PAGE and Western blot which was proceeded with P30 McAb produced by our laboratory. Results The size of amplified gene was 750 bp, the molecular weight of the expression protein was 30 ku. The expressed protein was futher identified by Western blot which was performed with P30 McAb. Conclusion The recombinant plasmid was successfully constructed, the protein of P30 gene of T. gondii over-expressed laid solid foundations for the diagnosis of toxoplasmosis as well as study of vaccines.
出处
《中国寄生虫病防治杂志》
CSCD
2005年第2期92-95,共4页
Chinese Journal of Parasitic Disease Control
基金
山东省科技厅资助项目。
