摘要
目的以pMIR-reporter为载体骨架,通过双酶切方法和同源重组方法构建载体,比较两种不同载体构建方法的优缺点。方法双酶切方法使用HindⅢ和MluⅠ两种限制性内切酶分别酶切pMIR-reporter和经T-A克隆后靶基因PCR片段,形成带有相同粘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感受态细胞,转化菌涂布含氨苄青霉素的固体培养基平板进行阳性单克隆筛选并测序。同源重组方法利用同源重组原理,通过重组酶将靶基因PCR片段和经HindⅢ酶切pMIR-reporter线性载体重组连接,连接产物转化入大肠埃希菌感受态细胞中,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。结果从载体构建耗时、总步骤数和阳性率三方面进行比较,双酶切载体构建方法需要26个独立试验,试验净耗时96h,阳性率为95%;同源重组法需要14个独立试验,试验净耗时33h,阳性率为70%。结论双酶切载体构建方法步骤繁琐。同源重组法步骤相对简单,可以高效快速地构建载体,但是假阳性率略高。
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 pMIR-reporter vectors. Methods In conventional double enzyme restriction, PCR fragments of the target gene were first inserted into a T-A cloning vector. The restriction enzymes HindⅢ and Mlu I were then used to generate "sticky ends" on a linearized pMIR- reporter vector and PCR fragments of a target gene from a T-A cloning vector. The fragments were ligated to form a functional pMlR-reporter vector with the aid of T4 ligation enzyme. In recombination, the target gene was inserted into the Hind HI-cleaved pMIR-reporter vector using a recombination enzyme. Both the ligated products were transformed into Escherichia coli and plated onto selective medium with ampicillin. Results To determine the advantages and disadvanta ges of both methods, conventional double enzyme restriction was compared to recombination in terms of the time course, the total number of steps, and the percentage of selected clones with the vector in question. Results indicated that conven- tional double enzyme restriction required 26 independent steps and took 96 hours in total. The vector in question was iden- tified in 95 %0 of selected clones. However, recombination required 14 independent steps and took 33 hours in total. The vector in question was identified in 70% of selected clones. Conclusion Compared to conventional double enzyme re striction, recombination involved simpler procedures and it allowed a vector to be constructed efficiently and quickly, but it also yielded a somewhat higher rate of false positives for clones containing the vector in question.
出处
《中国病原生物学杂志》
CSCD
北大核心
2015年第6期495-499,共5页
Journal of Pathogen Biology
关键词
载体
酶切
连接
同源重组
Vector
restriction enzymes
ligation
recombination