日本血吸虫卵壳蛋白前体基因Sj423的克隆表达及分析

据日本血吸虫菲律宾株编码卵壳前体蛋白的一段230bp的序列设计PCR引物,以日本血吸虫中国大陆株成虫mRNA为模板,用RT-PCR方法扩增出日本血吸虫中国大陆株相应的基因片段、然后根据测序结果设计一系列引物,用5’RACE和3’RACE法扩增出该基因cDNA的5’和3’端,再根据测序结果设计全长引物,用RT-PCR方法扩增出大小为423bp的片段。经序列分析推断该基因片段为编码日本血吸虫中国大陆株卵壳前体蛋白基因的完整阅读框,对相应基因组区段测序证实该基因没有内含子(GenBank登录号:AF519182)。将其克隆到表达载体pET28c(+)中,在大肠杆菌中获得表达,融合表达产物分子量约为20.9 kD。Real-ti me PCR结果显示该蛋白在尾蚴感染宿主后第23d即卵壳形成时高表达。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性。

Cloning and expression of Sj423 gene encoding the egg-shell precursor protein of Schistosoma japonicum

The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RTPCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S. japonicurn, the corresponding mDNA fragment of Chinese strain as template and then the 5′ and 3′ends of this gene eDNA were amplified with 5′RACE and 3′RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame （ORF） of gene encoding the egg-shell precursor protein of S. japonicum. （Chinese strain）. As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E. coli after cloning into the expression vector pET28c（＋）. The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S. japonicum;indicating the good antigenicity of this expressed product.