福氏志贺菌ipaC基因的克隆及序列分析*
Amplification and cloning of shigella flexneri ipaC gene
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摘要:目的 构建福氏志贺菌毒力蛋白基因(ipaC)重组表达质粒。方法 根据ipaC已知序列, 设计合成一对引物, 用PCR方法从福氏志贺菌毒力质粒上扩增出ipaC基因片段, 克隆到原核表达质粒pET32a中, 转化大肠埃希菌TG1感受态细胞, 经酶切和PCR鉴定, 然后进行测序。结果 ipaC基因体外扩增产物大小约为1112bp.重组质粒经双酶切和PCR鉴定表明为正确重组子, 测序结果与已知序列基本吻合。结论 在国内首次克隆了福氏志贺菌ipaC基因, 为下一步细菌性痢疾发病机制的研究奠定基础。Abstract:Objective To constract a recombinant plasmid containing Shigella.flexneri toxic protein gene(iFaC).Methods A couple of primers were designed for PCR according to the known sequence of ipaC.The ipaC gene obtained by amplification from plasmid DNA of S.flexneri by PCR technique was cloned into plasmid of pET 32a directionally.The recombinant plasmid pET 32a-ipaC was transferred into competent E.coli TG1.The recombinants were screened and identified by restriction analysis and PCR, the cloned gene was sequenced.Results The size of amplified ipaC gene was 1112 bp.The correct recombinant plasmid pET 32-aipaC was isolated and confir med by restriction analysis and PCR.DNA sequencing show ed the DNA sequence of the cloned gene was the same as the published sequence.Conclusion The ipaC gene was first successfully amplified and cloned into plasmid pET 32a in our country.It provided the basic material for studying the pathogenesis bacillary dysentery.
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