【摘要】 [目的 ]探索研制血吸虫病疫苗的新路径 ,对日本血吸虫线粒体相关蛋白进行基因克隆及特性鉴定。[方法 ]分析本室筛选日本血吸虫成虫cDNA文库获得的 1个cDNA片段 (Sj338 2 4)的开读框序列 ,在其上下游分别设计引物A和B ,并以该cDNA片段为模板进行PCR扩增后 ,将该片段重组于pGEM T中并进行DNA测序鉴定及检索。再经酶切后将该基因片段亚克隆入表达载体pGEX 6P 1,并进行蛋白表达、纯化及抗原性鉴定。 [结果 ]该目的基因PCR产物全长共 487bp ,其开读框由 45 9bp组成 ,编码 15 3个氨基酸经残基组成的多肽。DNA序列同源性分析发现Sj338克隆基因与人及褐鼠的线粒体外膜蛋白的部分编码基因较高度同源。重组质粒pPEX 6P 1 Sj338能高效融合表达 ,理论蛋白的分子量为 17kDa。SDS PAGE和Westernblotting检测结果表明 ,重组蛋白rSj338具有良好的抗原性。 [结论 ]Sj338可能为日本血吸虫线粒体相关蛋白的基因 ,重组蛋白有望成为新的疫苗候选分子。更多还原

【Abstract】 Objective] To subclone and characterize a cDNA clone coding for Schistosoma japonicum (S.j.) mitochondria-related protein. [Methods] The open reading frame of the fragment(Sj338/24) obtained from an adult worm cDNA library of S j. was analysed, at the upstream and downstream of the open reading frame(ORF) the primers A and B were designed,respectively, and the cDNA fragment was used as PCR template. The Sj338 gene fragment obtained was amplified by PCR method and then subcloned into pGEM-T vector for sequencing. The gene sequence was analyzed and the target fragment was restrictedly digested and subcloned into expression vector pGEX-6P-1. The expressed recombinant protein was purified and characterized. [Results] The cloned Sj338 gene was demonstrated to be 487 bp long containing one 459 bp ORF, encoding a protein with a molecular weight of 17 kDa.The nucleotide sequence of the cloned gene Sj338 had higher homology with those genes coding for mitochondrial outer membrane protein of Homo sapiens and Rattus norvegicus . The recombinant construct of pGEX-6P-1/Sj338 could be expressed efficiently and the antigenicity of its product rSj338 has been demonstrated by Western blotting. [Conclusion] Sj338 may be the gene coding for S j. mitochondria-related protein and the recombinant protein may be used as a new vaccine candidate .更多还原