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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。
Objective To explore a new path of schistosomiasis vaccine and to clone and characterize the mitochondria-associated protein of Schistosoma japonicum. Methods The open reading frame of a cDNA fragment (Sj338 / 24) obtained from the adult Schistosoma japonicum cDNA library was screened in our laboratory. Primers A and B were designed upstream and downstream of the cDNA library. After PCR amplification, The fragment was recombined in pGEM-T and verified by DNA sequencing. After digestion, the gene fragment was subcloned into the expression vector pGEX-6P-1, and the protein expression, purification and antigenicity were identified. Results The PCR product of the target gene was 487bp in length. The open reading frame consisted of 459bp and encoded a polypeptide of 153 amino acid residues. DNA sequence homology analysis found that Sj338 cloned gene and human and brown rat mitochondrial outer membrane protein partial coding gene more highly homologous. The recombinant plasmid pPEX-6P-1 / Sj338 could express efficiently and fusion protein with the molecular weight of 43kDa. SDS-PAGE and Western blot results showed that the recombinant protein rSj338 has good antigenicity. Conclusion · Sj338 may be the gene of Schistosoma japonicum related to mitochondria, and recombinant protein is expected to become a new vaccine candidate.