登革2型病毒全长cDNA克隆定点诱变的OL-PCR方法  

OL-PCR for site-directed mutagenesis of full-length cDNA clone of DEN-2

作　　者：赵卫[1] 胡志君[1] 杨佩英[1] 秦鄂德[1] 于曼[1] 陈水平[1] 王鹏程[1] 耿丽卿[1] 范宝昌[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病毒学专业实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2001年第6期662-665,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 30 0 0 0 14 4 );军事医学科学院创新基金资助项目

摘　　要：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　OLObjective To generate the oligonucleotide directed mutants of the full length cDNA clone of dengue 2 virus. Methods Two DNA fragments with single point mutation (E62 or E203) were amplified with four pairs of oligonucleotide primers by OL PCR and then cloned into pGEM T vectors respectively. The recombinant T vectors, T TB62 and T TB203, were digested with ClaⅠ+SphⅠand SphⅠ+NheⅠ, then ligated to pDVWS501 with T4 DNA ligase respectively. The recombinant plasmids, TB62 and TB203, were sequenced. Results The results of DNA sequencing indicated that TB62 and TB203 with point mutation were attained. Conclusion OL PCR is a kind of site directed mutagenesis method to achieve high mutation efficiency. [

关 键 词：登革2型病毒 CDNA克隆 定点诱变 OL-PCR 登革热病毒 

分 类 号：R346[医药卫生—基础医学]