弓形虫ROP18 基因原核表达载体构建及表达.docVIP

弓形虫 赵焕阁 周松林 黄用豪 郭峻莉 黄凤迎 林映莹 王华 谭光宏 （海南医学院海南省热带病重点实验室，海南 海口 571101） 摘 要：【目的】克隆编码刚地弓形虫ROP18基因,构建原核表达载体pET-ROP18进行体外表达。 【方法】克隆弓形虫ROP18基因，将其插入原核表达载体pET-28a中构建重组质粒pET-ROP18，转入大肠埃希菌BL21中IPTG诱导，SDS观察重组蛋白表达情况，Western-blot检测重组蛋白的反应原性。【结果】原核表达载体构建成功，重组蛋白在37℃1mmol/LIPTG诱导4h条件下表达量最大，Western-blot分析结果表明重组蛋白具有一定的反应原性。【结论】构建的原核表达载体成功，重组蛋白具有一定的反应原性。 关键词：弓形虫；ROP18；原核表达 Construction of prokaryotic expression vector and expression of Toxoplasma gondii ROP18gene ZHAO HUAN-GE ZHOU SONG-LIN HUANG YONG-HAO GUO JUN-LI HUANG FENG-YING LIN-YIN YING WANG-HUA TAN GUANG-HONG (Key Laboratory for Tropical Diseases, Hainan Medical College, Haikou 571101) Abstract：【Objective】To clone the ROP18 gene from Toxoplama gondii and construct prokaryotic expression vector pET-ROP18 to express on BL21 strain.【Methods】ROP18 gene fragment was amplified by PCR and insert into pET-28a vector to form the recombinant plasmid pET-ROP18,The recombinant plasmid pET-ROP18 was transformed into E.coliBL21 and induced with IPTG.The expression situation of recombinant protein was analysis by SDS. Its reactionogenicity was examined by western-blot analysis.【Results】The prokaryotic expression vector pET-ROP18 was successfully constructed, recombinant protein high level expression was found at 1mmol/L IPTG condition after incubation at 37℃ for 4h. Western-blot analysis show the recombinant protein has been expressed with reactionogenicity.【Conclusion】The prokaryotic expression vector pET-ROP18 was successfully constructed and the recombinant protein has been expressed with reactionogenicity Keywords：Toxoplasma gondii;rhoptry protein18 (ROP18); prokaryotic expression; 弓形虫是一种世界性分布的专性细胞内寄生原虫，可感染包括人类在内的所有哺乳动物，引起人兽共患寄生虫病。孕妇和免疫功能低下者感染弓形虫造成的危害尤为严重。近年来，，ROP18属于ROP2ROP18也是弓形虫体避免被宿主免疫系统杀死的一个屏障[3]，该抗原可用于弓形虫病诊断和作为疫苗的候选分子。本研究通过克隆弓形虫ROP18基因，构建原核表达载体，IPTG诱导，Western-blot检测，为进一步从分子水平研究其免疫原性和保护性奠定了基础。 1.材料与方法 1.1 虫株、菌种、质粒及细胞 弓形虫RH株DH5α、BL21(DE3)、原核表达载体pET-28a均由本实验室保存。TaqDNA聚合酶、限制性内切HindⅢ、BamHⅠ、T4PCR产物纯化试剂盒、胶回收试剂盒购自大连宝生物（TATARA）公司；