单核细胞增生性李斯特菌iap基因克隆和PCR条件优化

Cloning of iap gene of Listeria monocytogenes with PCR optimization

目的构建单核细胞增生性李斯特菌Listeriamonocytogenes54002—4株p60蛋白iap基因原核克隆载体。方法利用自行设计的引物通过梯度PCR优化扩增条件，扩增出单核细胞增生性李斯特菌54002—4株的iap基因。在iap基因的5’端和3’端分别引入BamHI和XhoI2个酶切位点，琼脂糖凝胶电泳分析、回收PCR产物，并将回收纯化的PCR产物与pMD18．T载体进行连接。将该重组质粒转化入大肠杆菌JMl09感受态细胞，经1mmol／LIPTG诱导4～6h后，观察转化效果。结果培养无色菌落细菌，提取质粒，经PCR鉴定和核苷酸序列测定后确定获得阳性重组质粒pMDl8-T—IaP。结论通过梯度PCR摸索出最佳反应条件，建立PCR优化条件和方法，经转化获得iap基因克隆载体。

Objective To construct a prokaryotic cloning vector of invasion- associated protein （iap） gene p60 of Listeria monocytogenes. Methods Under gradient PCR amplification conditions, Listeria monocytogenes 54002-4 strain iap gene was amplified with our in-house primers. BamH I and Xho I sites were induced to 5＇ and 3＇ terminals of iap gene, respectively. PCR products were then analyzed and collected by agarose gel electrophoresis. Purified PCR product was connected with the vector pMD18-T. The recombinant plasmid was transformed into Escherichia coli JM109 competent cells, with transformation effect observed after I mmol/L IPTG induction for 4-6 h. Results Colorless bacterial colony was cultured and verified to produce positive recombinant plasmid pMDl8-T-Iap by PCR and nucleotide sequencing. Conclusions PCR optimization conditions and methods are established through gradient PCR. Iap gene cloning vector is obtained after transformation.