弓形虫关键粘附蛋白在大肠埃希菌BL21(DE3)中的表达

Prokaryotic Expression of T.gondii Adhesion Protein in Escherichia coli BL21(DE3)

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 obtain the recombinant protein by cloning the rhoptry adhesion proteins -- Toxoplasma gondii ROP2 gcne, which was screened out from cDNA library, into pET -30a plaimide and thereby ex- pressed it in E. coli BL21 （DE3）. Methods Specific primers containing BamHI and HindIII restriction en- zyme site were designed. ROP2 gene was amplified by polymerase chain reaction （PCR） using T. gondii cDNA as a template. PCR products were purified and digested with the restriction enzymes BamHI and HindI- II, and then sub - coned into prokaryotic vector PMD - 18T and then into PET3Oa, and the recombinant pET -30a -rop2 was identified by PCR. After transforming into E. coli BL21 （DE3）, recombinant ROP2 protein was induced by isopropyl - D - thiogalactopyranoside （IPTG） and detected by SDS - polyacrylam-ide gel electrophoresis （SDS - PAGE ）. Results A fragment of 1223 bp, the same size as predicted, was obtained in PCR and double enzyme digestion. Recom- binant ROP2 was overexpressed in E. coli BL21 （DE3） with a molecular weight of 52kDa. Conclusion The PET- 30a- ROP2 could be constructed successful- ly and the recombinant ROP2 could be expressed effi- ciently in E. coli BL21 （DE3） under this experimentcondition, which provides a support for further research on the relation between T. gondii adhesion protein and enterocyte.