王敏敏;赵婕;常亚磊;陈浩男;陈思思;宋静;魏建宏;

• 1:山西医科大学汾阳学院基础医学部
• 2:山西医科大学汾阳学院医学检验系

摘要：

目的以PLVX-IRES-ZsGreen1为载体骨架,通过双酶切方法和无缝克隆方法构建HCV CORE真核表达载体,比较2种不同载体构建方法的优缺点,以期为合理选择载体构建方法提供依据。方法双酶切方法使用BamHⅠ和EcoRⅠ两种限制性内切酶分别酶切PLVX-IRES-ZsGreen1和目的基因聚合酶链反应(PCR)片段,形成带有相同黏性末端的线性载体片段和PCR片段,然后利用T4DNA连接酶连接,连接产物转化大肠埃希菌DH5α感受态细胞,转化菌涂在含氨苄青霉素的固体培养基中进行阳性单克隆筛选并测序。无缝克隆方法利用同源重组的原理,通过重组酶将靶基因PCR片段和经XhoⅠ酶切的PLVX-IRES-ZsGreen1线性载体重组连接,连接产物同样转化入感受态细胞中,转化菌涂布含氨苄青霉素的固体培养基平板进行阳性克隆筛选并测序。结果跟双酶切载体构建方法比较,同源重组法步骤相对简单,可以更高效快速地构建载体,但是假阳性率略高。结论同源重组法可更高效地构建载体,但假阳性率高。

关键词： 遗传载体;克隆,分子;同源重组

Abstract: Objective In molecular biology experiments,expression vectors are usually constructed in order to stably express foreign genes in cells.In this study,two different methodologies were used to generate HCV CORE vecto,for the more reasonable in selection of constructing vectors.Methods The restriction enzymes BamHⅠand EcoRⅠwere then used to generate " sticky ends" on a linearized PLVX-IRES-ZsGreen1 vector and PCR fragments of a target gene.The fragments were ligated to form a functional PLVX-IRES-ZsGreen1-CORE vector with the aid of T4 ligation enzyme.The ligated products were transformed into Escherichia coli DH5αcompetent cell and plated onto selective medium with ampicillin.In seamless cloning,utilizing the principle of homologous recombination,the PCR fragment of targeted gene was recombined with the XhoⅠ-cleaved PLVX-IRES-ZsGreen1 vector of the target gene by the recombinant enzyme.Then all products were treated with the same conditions.Results Compared to conventional double enzyme restriction,Seamless cloning involved simpler procedures and it allowed a vector to be constructed efficiently and quickly,but it also yielded a higher rate of false positives for clones containing the vector in question.Conclusion Seamless cloning can construct a vector efficiently and quickly,but it have a high rate of false positives.

Keywords: Vector construction;Cloning;molecular;Homologous recombination

基金项目: 山西省大学生创新创业训练计划项目(2014052)

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