刚地弓形虫致密颗粒蛋白GRA7基因的重组、表达及鉴定  

Cloning and expression of Toxoplasma gondii GRA7 gene in Escherichia coli

作　　者：张成芳[1,2,3] 李瑾[1] 张海国[3] 魏庆宽[1,2] 王卫艳[1,2] 肖婷[1,2] 徐超[1] 贾凤菊[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 [3]山东省济宁市第一人民医院

出　　处：《中国病原生物学杂志》2014年第3期245-248,共4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)

摘　　要：目的构建弓形虫致密颗粒蛋白7(GRA7)基因重组质粒并在大肠埃希菌中进行表达。方法根据GRA7基因序列设计合成引物,用PCR方法从弓形虫基因组DNA中扩增GRA7基因片段,再克隆到pGEX-4T载体中,重组质粒经酶切、PCR鉴定并测序;重组质粒在大肠埃希菌BL21(DE3)中诱导表达,表达产物经SDS-PAGE分析并纯化,Western blot分析其反应原性。结果 GRA7基因PCR产物大小约为714bp,与预期相符;重组质粒经酶切及PCR鉴定构建成功,测序结果与已知序列吻合;重组质粒转化菌经IPTG诱导后表达的GRA7融合蛋白分子质量单位约为53ku,该蛋白可被His标签抗体识别。结论成功重组了弓形虫GRA7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。Objective To construct a recombinant plasmid containing the dense granule protein （GRA7） gene of Toxoplasma gondii and express it in Escherichia coll. Methods The truncated GRA7 gene was amplified from the genomic DNA of the T. gondii RH strain and cloned into the plasmid pGEX-4T. Expression of the recombinant pGEX-4T-GRA7 was induced in BL21 （DE3） with IPTG. The expressed proteins were analyzed using SDS-PAGE and purified and then subjected to Western blotting. Results The amplified GRA7 gene was about 714 bp in length, and the gene was confirmed using DNA sequencing. The recombinant plasmid was verified to be correct according to PCR and double restriction enzyme digestion. SDS-PAGE and Western blotting indicated a positive reaction band at about 53 ku. The protein was recognized by His-tag antibody. Conclusion The truncated GRA7 gene of T. gondii has been successfully cloned and expressed in BL21 （DE3） cells.

关 键 词：弓形虫 致密颗粒蛋白7 融合蛋白 蛋白纯化 

分 类 号：R382.5[医药卫生—医学寄生虫学]