日本血吸虫再感染相关蛋白基因的克隆和原核表达

目的　从日本血吸虫 (S .japonicum )尾蚴文库扩增S .japonicum再感染相关蛋白 (RIRP)基因 ,进行克隆并原核表达出RIRP ,为进一步的功能研究奠定基础。　方法　根据已登陆的S .japonicum成虫期RIRP基因的序列设计两条特异引物 ,以S .japonicum尾蚴cDNA文库为模板扩增RIRP基因cDNA ,将该基因的cDNA克隆入原核表达载体 pGEX 4T 1和真核载体 pcD NA3 .1。转化的克隆通过氨苄青霉素的筛选、PCR扩增、双酶切和最后的测序鉴定。重组的 pGEX 4T 1/RIRP在大肠埃希菌BL2 1(DE3 )中进行表达。SDS PAGE鉴定其在大肠埃希菌中的表达情况和分子质量。用抗 2 6kuGST单抗作western杂交进一步鉴定融合蛋白的表达。　结果　从S .japonicum尾蚴文库中扩增出一条长为 462bp的cDNA片段 ,经测序证实其为RIRP基因的全长cDNA。它编码含 15 3个氨基酸的蛋白质—RIRP ,预测其分子质量单位为 17.5 45ku。RIRPcDNA已被成功克隆入 pGEX 4T 1和pcDNA3 .1载体 ,大肠埃希菌BL2 1(DE3 )编码一个约 17ku的蛋白质。　结论　RIRP基因首次从S .japonicum尾蚴文库中扩增出来 ,并成功构建了原核和真核表达载体 ,而且 ,它首次由大肠埃希菌表达 ,表达蛋白的分子质量单位约 17ku。

CLONING AND EXPRESSION IN ESCHERICHIA COLI OF SCHISTOSOMA JAPONICUM REINFECTION RELATED PROTEIN GENE cDNA

Objective To amplify reinfection related protein(RIRP) gene cDNA of Schistosoma japonicum ,clone it into prokaryotic and eukaryotic expression plasmids,and express it in Escherichia coli . Methods A pair of primers was designed according to the known RIRP gene cDNA of S. japonicum adult worm. This gene was amplified by PCR from S. japonicum cercaria cDNA library. Then,it was cloned into the prokaryotic vector pGEX-4T-1 and eukaryotic vector pcDNA3.1. The transformed clones were screened and identified by PCR amplification,restriction endonuclease analysis and sequencing. The recombinant pGEX-4T-1/RIRP was expressed in E. coli named BL21(DE3). SDS-PAGE was used to identify whether RIRP was expressed and its molecular weight. Anti-26 ku GST monoclonic antibody was used to identify the fusion protein by western blot. Results A 462 bp length cDNA was amplified from S. japonicum cercaria cDNA library and identified to be RIRP gene cDNA by sequence analysis. The open reading frame(ORF) of the gene encodes a protein of 153 amino acids. The predicted molecular weight of the protein is 17.545 ku. Its cDNA was successfully cloned into the pGEX-4T-1 vector and pcDNA3.1,and then,it was expressed in BL21(DE3) with molecular weight of about 17 ku. Conclusion The RIRP gene cDNA of S. japonicum was firstly PCR amplified from S. japonicum cercaria cDNA library. Its recombinant prokaryotic and eukaryotic expression plasmids were successfully constructed and its nucleotide sequence was determined. It was firstly expressed in E. coli with MW of about 17 ku.