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目的 对带有登革 2型病毒 (DEN 2 )全长cDNA的质粒pDVWS5 0 1上E6 2、E2 0 3位点进行定点诱变。方法 设计 4对点诱变引物 ,运用OL PCR(overlapPCR)法 ,扩增出分别在E6 2或E2 0 3位带有点突变的 2条DNA片段 ,克隆至T载体 ,获T TB6 2、T TB2 0 3两个克隆。将T TB6 2用ClaⅠ和SphⅠ分别酶切 ,T TB2 0 3用SphⅠ +NheⅠ酶切后 ,用T4连接酶分别连接至pDVWS5 0 1,获重组质粒TB6 2和TB2 0 3。对TB6 2和TB2 0 3进行序列测定。结果 成功得到分别在E6 2、E2 0 3位带有点突变的TB6 2、TB2 0 3克隆。结论 OL PCR法是具有较高突变效率的定点诱变方法
Objective To carry out site-directed mutagenesis on the E6 2 and E2 0 3 sites of plasmid pDVWS5 0 1 with the full-length cDNA of Dengue 2 virus (DEN 2). Methods Four pairs of point mutagenesis primers were designed. Two DNA fragments with point mutations at E6 2 or E2 0 3 were amplified by OLPCR (overlap PCR) and cloned into T vector. T TB6 2, T TB2 0 3 Two clones. T TB6 2 was digested with ClaI and Sph I, respectively. T TB2 0 3 was digested with SphI + NheI and ligated into pDVWS5 0 1 with T4 ligase respectively to obtain recombinant plasmids TB6 2 and TB2 0 3. TB6 2 and TB2 0 3 were sequenced. Results TB6 2 and TB2 0 3 clones with point mutations at E6 2 and E2 0 3 were successfully obtained. Conclusion OL PCR method is a site-directed mutagenesis method with higher mutation efficiency