日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究(英文)  

Cloning,Expression and Immunization of The Hypoxanthine-guanine Phosphoribosyltransferase for Schistosoma japonicum Chinese Strain

作　　者：余俊龙[1] 汪世平[1] 何卓[1] 戴橄[1] 李文凯[1] 姜孝新[1] 曾少华[1] 肖小芹[1] 徐绍锐[1] 吕志跃[1] 彭先楚[1] 周松华[1] 刘雪琴[1] 

机构地区：[1]中南大学湘雅医学院血吸虫病研究室,长沙410078

出　　处：《生物化学与生物物理进展》2006年第7期665-672,共8页Progress In Biochemistry and Biophysics

基　　金：国家"十五"重大科技专项(2002AA2Z3343);国家高技术研究发展计划(863)(2004AA2Z3530);湖南省"十一五"重大专项;湖南省重点学科建设专项经费联合资助(03-985-3-7)~~

摘　　要：根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)EST(BU803192)以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA(1270bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶(SmHGPRT)全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%.将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28ku.用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带.重组蛋白动物免疫保护性结果显示:在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性(P<0.05,P<0.01).结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶(SjHGPRT)全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.A 1 270 bp full-length cDNA fragment was obtained from the Schistosornajaponicum （Chinese strain） adult cDNA library after the 3＇ and 5＇ ends of the incomplete expression sequence tag （EST） of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum （SjHGPRT） were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library γgt11 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-gnanine phosphoribosyltransferase of Schistosorna mansoni （SmHGPRT）, contained a complete open reading frame（ORF）. The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coll. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosomajaponicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram （LEPG）, fecal eggs per gram （FEPG） and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjTIGPRT fulMength cDNA can be cloned and expressed in E.coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partial protection vaccine candidate.

关 键 词：日本血吸虫 次黄嘌呤鸟嘌呤磷酸核糖转移酶 基因克隆 基因表达 

分 类 号：R383.24[医药卫生—医学寄生虫学]