日本血吸虫p50亲免素基因的扩增及其免疫学特性

目的扩增日本血吸虫(Sj)p50亲免素(IP)基因全长cDNA和DNA序列,进行原核克隆和研究其免疫学特性。方法从Sj成虫RNA中RT-PCR扩增Sjp50亲免素基因完整的编码阅读框,从Sj成虫基因组中扩增其DNA序列,对其cDNA序列和DNA序列进行比较分析,寻找内含子。将其cDNA序列克隆入pET30a( )体中,原核表达并纯化表达产物,Western-blot比较重组蛋白与Sj体内相应天然蛋白的免疫原性和免疫反应性,间接免疫荧光法对p50亲免素进行组织定位。结果Sjp50IP基因DNA序列与cDNA相比,有6个内含子;重组p50IP与Sj体内的天然蛋白具有相同的免疫学特性,该蛋白位于Sj成虫的被膜上。结论成功扩增得到了Sjp50IP基因的全长编码区的cDNA序列和DNA序列,其DNA序列中有6个内含子;重组Sjp50IP的免疫学特性支持将该基因作为疫苗进行进一步的研究。

Cloning and Immunological Characterization of Schistosoma japonicum p50 Immunophilin Gene

Objective To clone and study the immunological characterization of Schistosoma japonicum (Sj) p50 immunophilin gene. Methods The full-length cDNA of p50IP gene was obtained by RT-PCR from Sj adult worm RNA and the gene was amplified by PCR from Sj genomic DNA. The p50IP cDNA was cloned into pET30a( )vector and the recombinant p50IP protein was expressed in E.coli. The expressed products were purified by affinity chromatography column. Western-blot was used to identify the recombinant protein's immunological characterization. The indirect immunofluorescence assay was used to locate expression of p50IP in the tissue of Sj adult worm. Results The Sjp50IP cDNA and gene were obtained. The Sjp50IP gene contains 6 introns. The recombinant p50IP has the same immunological character as the native one in parasite. It was localized in the tegument. Conclusions Sjp50IP gene and cDNA were successfully amplified and cloned. The immunological characterization of the recombinant p50IP will give the basis for further studies especially the development as a vaccine candidate.