CAJ | 학술논문

目的构建单核细胞增生性李斯特菌Listeria monocytogenes 54002-4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BamH Ⅰ和Xho Ⅰ 2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pMD 18-T载体进行连接.将该重组质粒转化人大肠杆菌JM109感受态细胞,经1 mmol/L IPTG诱导4～6 h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pMD18-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.
목적 구건단핵세포증생성리사특균Listeria monocytogenes 54002-4주p60단백iap기인원핵극륭재체.방법 이용자행설계적인물통과제도PCR우화확증조건,확증출단핵세포증생성리사특균54002-4주적iap기인.재iap기인적5'단화3'단분별인입BamH Ⅰ화Xho Ⅰ 2개매절위점,경지당응효전영분석、회수PCR산물,병장회수순화적PCR산물여pMD 18-T재체진행련접.장해중조질립전화인대장간균JM109감수태세포,경1 mmol/L IPTG유도4～6 h후,관찰전화효과.결과 배양무색균락세균,제취질립,경PCR감정화핵감산서렬측정후학정획득양성중조질립pMD18-T-Iap.결론 통과제도PCR모색출최가반응조건,건립PCR우화조건화방법,경전화획득iap기인극륭재체.
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 1mmol/L IPTG induction for 4-6 h. Results Colorless bacterial colony was cultured and verified to produce positive recombinant plasmid pMD18-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.