Sha Dan, Tan Feng, Pan Chang-Wang, Liang Shao-Hui
Wenzhou Medical College, Wenzhou 325035, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2007 Dec;25(6):447-50.
To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase (NTPase) gene of Toxoplasma gondii, and analyze its antigenicity.
NTPase 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 electrophoresis (SDS-PAGE) and Western blotting.
NTPase-II gene was specifically amplified, and the homology of DNA sequence was 100% to that in the GenBank. 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.
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.
克隆并表达弓形虫核苷三磷酸水解酶（NTPase）基因的原核重组质粒，并分析其抗原性。
采用聚合酶链反应（PCR）从弓形虫RH株中扩增NTPase基因，并克隆至pGEM-T Easy载体。通过BglII、HindIII酶切及测序对阳性克隆进行筛选和鉴定。随后将目的基因亚克隆至原核表达载体pBAD-HisB，并转化至大肠杆菌BL21（DE3）。用Ni-NTA琼脂糖纯化表达的重组蛋白，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法进一步分析。
特异性扩增出NTPase-II基因，其DNA序列与GenBank中的同源性为100%。SDS-PAGE显示，分子量正确的重组NTPase蛋白在大肠杆菌BL21（DE3）中高表达。蛋白质免疫印迹法证实，纯化的重组蛋白能被弓形虫免疫小鼠血清及小鼠抗重组蛋白血清特异性识别。
NTPase-II基因已在大肠杆菌BL21（DE3）中克隆并表达，纯化的NTPase-II基因蛋白具有特异性抗原性。
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