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目的构建弗氏志贺菌侵袭性基因ipaC诱饵质粒,并检测其自激活作用。方法 PCR法扩增弗氏志贺菌侵袭素ipaC基因,克隆入诱饵载体pSos,构建诱饵质粒pSos-IpaC,转化酵母菌cdc25H,进行Western blot分析,并检测诱饵质粒的自激活特性。结果酵母双杂交诱饵质粒pSos-IpaC经酶切及测序证明构建正确;Western blot分析显示,诱饵质粒表达了相对分子质量约214000的融合蛋白;诱饵质粒对酵母菌cdc25H无自激活作用,且该质粒表达的融合蛋白在宿主细胞质中定位正确。结论已成功构建了诱饵质粒pSos-IpaC,该质粒对酵母菌cdc25H无自激活作用。
Objective To construct the ipaC bait plasmid with Shigella flexneri invasion and to detect its self-activation. Methods The ipaC gene of Shigella flexneri was amplified by polymerase chain reaction (PCR) and cloned into bait vector pSos. The bait vector pSos-IpaC was constructed and transformed into Saccharomyces cerevisiae cdc25H. Western blot analysis and self-activation of bait plasmid were also performed. Results The bait plasmid pSos-IpaC was confirmed by restriction enzyme digestion and sequencing. Western blot analysis showed that the bait plasmid expressed a fusion protein with a relative molecular mass of about 214000. The bait plasmid had no self-activation activity on yeast cdc25H, and the plasmid The expressed fusion protein is correctly located in the host cytoplasm. Conclusion The bait plasmid pSos-IpaC has been successfully constructed, which has no self-activation activity on yeast cdc25H.