目的获得日本血吸虫病新的候选疫苗分子。方法用对日本血吸虫具有天然抗性的东方田鼠血清免疫筛选日本血吸虫肝期童虫cDNA文库,阳性克隆转入E.coliBM25.8环化成质粒,抽提质粒DNA,EcoRⅠ和Hind Ⅲ双酶切,琼脂糖凝胶电泳鉴定,插入片段进行核苷酸序列测序,并进行生物信息学分析。结果共获得12个不同的基因,插入片段长度为300~1100bp,其中2个具有完整的开放阅读框,为日本血吸虫新基因,命名为sj—sMf1和sj—sMf2,分别编码93个和61个氨基酸。前者含有cAMP磷酸化位点和酪蛋白激酶Ⅱ磷酸化位点各1个,后者含有1个酪蛋白激酶Ⅱ磷酸化位点和2个蛋白激酶c磷酸化位点。结论用东方田鼠血清作为探针筛选到2个日本血吸虫新基因。
Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoR Ⅰ and Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1 100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.