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目的:构建并鉴定pMDl8-T-ipaH重组质粒载体,为探索采用二聚体蝎形探针荧光更精确的定量检测志贺菌标准品的方法奠定基础。方法:以志贺菌袭性相关位的ipaH基因为模板,采用PCR进行扩增,电泳回收纯化,经酶切后克隆入载体质粒pMDl8-T中,最终构建重组质粒pMDl8-T-ipaH,进行酶切和测序鉴定。结果:成功克隆出151 bp左右的ipaH基因片段,导入质粒后,经检酶切及测序鉴定质粒基因片段与目标基因序列一致。结论:成功的构建了pMDl8-T-ipaH重组质粒,为进一步检测志贺菌建立标准品奠定了前期基础。
OBJECTIVE: To construct and identify pMDl8-T-ipaH recombinant plasmid vector and lay a foundation for exploring a more accurate and quantitative method for the quantitative determination of Shigella standards by using dimeric Scorpion probe. Methods: The ipaH gene of Shigella spp. Related gene was amplified by PCR and purified by electrophoresis. The recombinant plasmid pMD18-T-ipaH was cloned into the vector pMD18-T after digestion. The recombinant plasmid pMDl8-T-ipaH Digestion and sequencing identification. Results: The ipaH gene fragment of about 151 bp was successfully cloned. After being introduced into the plasmid, the plasmid gene fragment was identified by the restriction enzyme digestion and sequencing and the target gene sequence was consistent. Conclusion: The recombinant plasmid pMDl8-T-ipaH was successfully constructed, which laid the foundation for the further determination of Shigella to establish standard.