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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.
The primers were designed according to the nucleotide sequence of the λgt11 polyclonal site of Schistosoma japonicum cDNA library vector of Schistosoma japonicum phosphoglucuronosyltransferase (HGPRT) EST (BU803192) in the gene bank and the cDNA library of Schistosoma japonicum adult as template , The incomplete 3 ’end and 5’ end of SjHGPRT gene were amplified by anchor PCR and sequenced. The full length cDNA of SjHGPRT (1270 bp) was obtained by splicing by electronic software. Sequence analysis revealed that the fragment contained SjHGPRT gene In the complete reading frame, the nucleotide sequence of the full-length coding gene was 82% identical to the full-length coding gene of Schistosoma mansoni Hypoxanthine guanine phosphoribosyltransferase (SmHGPRT), and its theoretical deduced amino acid composition was similar to that of the Schistosoma mansoni hypoxanthine guanine Phosphotransferase identity was about 83% .The gene encoding cloned into the expression vector pQE30 in E. coli M15 to obtain accurate and efficient expression of the molecular weight of the expression product about 28ku.Antigen antigen Schistosoma japonicum immune serum pairs The expression product was detected by Western blot, and the obvious recognition band appeared at the predicted position.Immunoprotective results of recombinant protein animal showed that at (P <0.05, P <0.01) .The results showed that there was significant difference between the vaccine group and the control group (P <0.05, P <0.01) The full-length cDNA of SjHGPRT was successfully cloned and expressed in coliforms. The expressed product has good antigenicity and animal immunoprotective effect, and is a potential vaccine candidate with partial immunopotency against schistosomiasis .