弓形虫GRA6基因的重组、表达及鉴定  

Cloning and expression of the GRA6 gene of Toxoplasma gondii in Escherichia coli

作　　者：李瑾[1] 魏庆宽[1] 贾凤菊[1] 徐超[1] 肖婷[1] 王卫艳[1,2] 尹昆[1] 刘功振[1] 黄炳成 

机构地区：[1]山东省医学科学院,山东省寄生虫病防治研究所,山东济宁272033 [2]济南大学,山东省医学科学院医学与生命科学学院

出　　处：《中国病原生物学杂志》2015年第3期239-246,260,共8页Journal of Pathogen Biology

基　　金：山东省自然科学基金项目(No.2009C03083);山东省医药卫生科技计划项目(No.2011HW049,2014WS0330)

摘　　要：目的构建弓形虫致密颗粒蛋白6(GRA6)基因重组质粒并在大肠埃希菌中进行表达。方法根据弓形虫GRA6基因序列设计并合成引物,用PCR方法从弓形虫基因组DNA中扩增GRA6基因片段,扩增产物经1.0%琼脂糖凝胶电泳鉴定后,再克隆到pGEX-4T载体中,重组质粒经限制性内切酶BamHI、EcoRI酶切鉴定并测序后,将重组质粒转化大肠埃希菌BL21后并用IPTG诱导表达,产物经SDS-PAGE分析并纯化,以Western blot分析其反应原性。结果以弓形虫DNA为模板扩增GRA6基因片段,经1%琼脂糖凝胶电泳鉴定扩增产物大小为693bp,与理论值相符;重组质粒经酶切鉴定构建成功,测序结果与GenBank上弓形虫RH株GRA6序列AF239283.1同源性100%;重组质粒转化菌经IPTG诱导后表达的GRA6融合蛋白分子质量单位约为52ku,与GST-GRA6的理论分子质量相符,该融合蛋白可被GST标签抗体识别。结论成功重组了弓形虫GRA6基因,表达蛋白具有反应原性,为弓形虫诊断抗原试剂盒的制备奠定了基础。Objectives The purposes of this study were to construct a recombinant plasmid containing the granule protein 6（GRA6）gene of Toxoplasma gondii and to express it in Escherichia coli. Methods The ORF of GRA6 gene was amplified from the genomic DNA of the RH strain of T.gondii and cloned into a pGEX-4Tvector after digestion with BamHI and EcoRI.The recombinant pGEX-4T-GRA6 was then sequenced and transformed into E.coli BL21 competent cells,and protein expression was induced with IPTG.The expressed product was purified using GST resin and was then analyzed using SDS-PAGE.In order to evaluate the immunogenicity of the recombinant protein,the product was analyzed using Western blotting. Results The amplified GRA6 gene was about 693 bp in length,which was in accordance with expectations.The recombinant plasmid pGEX-4T-GRA6 was verified by double digestion with the restriction enzymes BamHI and EcoRI,and the nt sequence of GRA6 had 100%similarity to that of AF239283.1.SDS-PAGE results indicated that the GRA6 fusion protein had a molecular weight of about 52 ku.Western blotting indicated that GRA6 fusion protein was recognized by GST-tag antibodies. Conclusions The GRA6 gene of T.gondii was successfully cloned and expressed in BL21 cells.The GRA6 recombinant protein has immunogenicity and paves the way for preparation of diagnostic kits to detect T.gondii antigens.

关 键 词：弓形虫 致密颗粒蛋白6 基因克隆 融合蛋白 蛋白纯化 

分 类 号：R382.5[医药卫生—医学寄生虫学]