日本血吸虫(中国大陆株)14-3-3信号转导蛋白epsilon亚型基因[Parasitol1]真核表达重组质粒的构建及序列分析

Construction of eukaryotic expression recombinant plasmid and sequence analysis of 14-3-3 protein epsilon isoforms gene from Schistosoma japonicum(Chinese strain)

目的 构建日本血吸虫中国大陆株 14 - 3- 3信号转导蛋白epsilon亚型基因真核表达重组质粒 pBK -Sj14 - 3- 3,为进一步对重组蛋白的融合表达及保护性免疫的研究提供条件。方法 根据日本血吸虫 14 - 3- 3蛋白的核苷酸序列 ,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 ,用RT -PCR法合成日本血吸虫中国大陆株 14 - 3- 3蛋白epsilon亚型基因cDNA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 -Sj14 - 3- 3,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 RT -PCR产物、pGEM -T -Sj14 - 3- 3及 pBK -Sj14 - 3- 3分别经双酶切均获得一特异性基因片段 经测序分析后该片段具有一个 75 3bp完整开放阅读框 (openreadingframe ,ORF) ,由此推导的氨基酸序列具有多种蛋白激酶的磷酸化位点。 结论 成功地构建了日本血吸虫中国大陆株 14 - 3- 3信号蛋白epsilon亚型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。

To construct an eukaryotic expression recombinant plasmid pBK-Sj14-3-3 of 14-3-3 signal transduction protein(epsilon isoforms) gene from Schistosoma japonicum(Sj chinese strain) ,a pair of primers were designed and synthesized according to the known nucleotide sequence of Sj14-3-3 protein.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,a recombinant pBK-Sj14-3-3 was constructed and transferred into E.coli BL21.The nucleotide sequence was identified by endonuclease digestion.The same specific gene fragment was obtained by RT-PCR,endonuclease digestion of pGEM-T-Sj14-3-3 and pBK-Sj14-3-3.After sequencing,the fragment posses a complete open reading frame(ORF) containing 753bp,the deduced amino acid sequence posses varied phosphoric acidifying sites of protein kinases.It concludes an eukaryotic expression recombinant plasmid pBK-Sj14-3-3 of 14-3-3 signal transduction protein ( epsilon isoforms ) from Sj Chinese strain has been successfully constructed.Meanwhile,the nucleotide sequence and phosphoric acidifying sites of protein kinases of amino acid were particular analyzed.