摘要
目的对刚地弓形虫三磷酸核苷水解酶基因(NTPase)进行克隆、表达和鉴定。方法采用PCR扩增刚地弓形虫RH株的NTPase基因,克隆入pGEM-TEasy载体,经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB,并转入大肠埃希菌(E.coli)BL21(DE3)中进行诱导表达。用镍-次氮基三乙酸亲和层析柱纯化重组质粒pBAD-HisB-NTPase表达产生的含组氨酸的重组蛋白,用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析蛋白表达产物。结果PCR扩增得到特异的刚地弓形虫NTPase-Ⅱ基因序列,经测序鉴定无基因突变。SDS-PAGE结果表明NTPase-Ⅱ基因在E.coliBL21(DE3)中获得高效表达,其融合蛋白相对分子质量(Mr)约70000,与理论值相近。Western blotting分析结果显示,纯化的重组蛋白可被弓形虫感染的鼠血清及鼠抗重组蛋白血清识别。结论所克隆表达的弓形虫NTPase-Ⅱ重组蛋白具有良好的抗原性。
Objective To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase （NTPatse） gene of Toxoplasma gondii, and analyze its antigenicity. Method NTPatse gene was amplified by PCR from RH strain of T. gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglⅡ, HindⅢ digestion and sequenced. The target gene was then subcloned into prokaryotic expression vector pBAD-HisB and transformed into E. coli BL21 （DE3）. The expressed recombinant protein was purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and Western blotting. Results NTPase-Ⅱ gene was specifically amplified, and the homology of DNA sequence was 100% to that in the C, enBank. SDS- PAGE showed that the recombinant NTPase protein with correct molecular weight was expressed highly in E.coli BL21 （DE3）. Western blotting testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein serum. Conclusion The NTPase-Ⅱ gene has been cloned and expressed in E.coli BL21（DE3）, and the purified protein of NTPase-Ⅱ gene displays a specific antigenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2007年第6期447-450,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
浙江省科技计划项目基金(No.2006C33025)~~
关键词
弓形虫
三磷酸核苷水解酶基因
融合蛋白
抗原性
Toxoplasma gondii
Nucleoside tdphosphate hydrolase （NTPase）
Expression
Antigenicity
