日本血吸虫线粒体相关蛋白的基因克隆及特性鉴定

目的为探索血吸虫病疫苗的新路径,对日本血吸虫线粒体相关蛋白的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文库获得的一cDNA片段(Sj338/24)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459bp组成,编码153个氨基酸残基组成的多肽.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.重组质料pPEX-6P-1/Sj338能高效融合表达,融合蛋白的分子量为43kDa.SDS-PAGE和Western blot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侯选分子.

GENE CLONING AND CHARACTERIZATION OF MITOCHONDRIA RELATED PROTEIN OF SCHISTOSOMA JAPONICUM

Objective To subclone and character a cDNA clone coding for S.japonicum (Sj) mitochondriarelated protein. Methods To analyse the open reading frame of the fragment (Sj 338/24) which was obtained from an adult worm cDNA library of Sj, the primers A and B were designed respectively at the upstream and downstream of the open reading frame(ORF), and the cDNA fragment was used as PCR template. The Sj338 gene fragment was obtained and amplified by PCR method, 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 purifird and characterized. Result The cloned Sj338 gene was demonstrated to be 487bp containing one 459bp ORF, encoding a protein with a molecular weight of 17kDa. 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 recombimant construct of pGEX-6P-1/Sj338 could be expressed efficiently and the antigenicity of its product rSj338 has been demonstrated by Western Blot. Conclusion Sj338 may be the gene coding for Sj mitochondria-related protein and the recombinant protein may be used as a new vaccine candidate.