日本血吸虫(大陆株)26ku谷胱甘肽转移酶基因真核表达载体构建及序列分析

目的　构建日本血吸虫大陆株 2 6ku谷胱甘肽转移酶 (GST)基因真核表达质粒 pBK SjGST并进行序列分析 ,为进一步进行重组蛋白的表达及保护性免疫研究提供条件。方法　根据日本血吸虫菲律宾株 2 6kuGST核苷酸序列 ,设计合成一对引物 ,以日本血吸虫中国大陆株成虫总RNA为模板 ,通过RT PCR合成日本血吸虫中国大陆株 2 6kuGSTcDNA片段。将其克隆入 pGEM T载体 ,经双酶切及PCR鉴定后 ,再亚克隆入 pBK CMV真核表达质粒 ,构建重组质粒pBK SjGST ,转化到大肠杆菌BL2 1感受态细胞 ,提取重组质粒双酶切鉴定并进行序列分析。结果　PCR、pGEM T SjGST及 pBK SjGST分别经双酶切获得一特异性基因片段 .经测序分析该片段具有一个 670bp的全基因序列 ,而其开放阅读框 (openreadingframe,ORF)为 65 7bp ,编码 2 18个氨基酸 ,并对其基因序列及编码的蛋白质进行了分析。结论　成功地构建了日本血吸虫中国大陆株 2 6kuGST基因真核表达重组质粒 ,并对其核苷酸序列及推导的氨基酸序列蛋白激酶磷酸化位点进行分析

Construction of eukaryotic expression recombinant plasmid and sequence analysis of glutathione-S-transferase gene from schistosoma japonicum(Chinese Strain)

Objective To construct an eukaryotic expression recombinant plasmid pBK SjGST of glutathione S transferase gene from Schistosoma japonicum(Sj Chinese strain) and sequence analysis for further study of protein expression and protective immunity. Methods A pair of primers were designed and synthesized according to the known nucleotide sequence of Sj26 ku GST(Philippine strain). Using adult worms total RNA of Sj as template,a SjcDNA first strand synthesis was driven by RT PCR technique. The product was cloned into pGEM T vector and identified by double endonuclease digestion and PCR.The fragment was subcloned into an eukaryotic expression vector pBK CMV again,and a recombinant pBK SjGST was constructed and transferred into E.coli BL21.The nucleotide sequence was identified by endonuclease digestion. Results The same specific gene fragment was obtained by RT PCR,endonuclease digestion of pGEM T SjGST and pBK SjGST.Sequence analysis revealed that the fragment possessed a complete open reading frame(ORF) containing 657bp;the deduced 218 amino acid sequence possessed varied phosphoric acidifying sites of protein kinases. Conclusion An eukaryotic expression recombinant plasmid pBK SjGST of glutathione S transferase from Sj Chinese strain was successfully constructed. Meanwhile,the nucleotide sequence and phosphoric acidifying sites of protein kinases of amino acid were particularly analyzed.