简化的两步法细菌内同源重组高效制备腺病毒质粒  

Recombinant Adenovirus Plasmid Constructed by Simplied and High Performance Two-step Homologous Recombination in Bacteria

作　　者：陈建发[1] 黄宗海[1] 黄元媛[1] 汤福祥[1] 杨文宇[1] 车小燕[2] 

机构地区：[1]第一军医大学珠江医院普外科,广东广州510282 [2]第一军医大学珠江医院分子免疫研究室,广东广州510282

出　　处：《肿瘤防治杂志》2003年第5期473-475,共3页China Journal of Cancer Prevention and Treatment

基　　金：国家"八六三"计划生物和现代农业技术研究基金资助项目 (2 0 0 1AA2 1 71 71 );广东省自然科学基金重点项目(0 1 30 72 )

摘　　要：目的 :应用简化的两步法细菌内同源重组高效制备腺病毒质粒。方法 :对细菌内同源重组法进行改进和简化 ,先构建含腺病毒基因组质粒pAdEasy 1的BJ5 183细菌 ,筛选出链霉素和氨苄青霉素抗性菌落 ,继而应用氯化钙法制作BJ5 183pAdEasy 1感受态细菌。用PmeⅠ酶使转移质粒pAdtrack CMV TK线性化 ,和BJ5 183pAdEasy 1感受态细菌混合进行转化 ,在含 12 5 μg mL卡那霉素的LB琼脂平皿上培养 ,筛选出卡那霉素抗性的细菌进行质粒抽提纯化 ,获得重组腺病毒质粒。结果 :卡那霉素抗性细菌有 2种 ,一种是含pAdeasy CMV TK(约 34kb) ,另一种含pAdtrack CMV TK(约 10kb) ,两者可经琼脂糖电泳加以鉴别。构建重组腺病毒质粒的成功率达 90 % (9 10 )。结论 :简化的两步法细菌内同源重组是一种简便易行。Objective To construct a recombinant adenovirus plasmid by a simplied and efficient two step homologous recombination in bacteria.Methods First,pAdEasy 1 was transformed into E.coli BJ5183 to generate the so called BJ5183pAdEasy 1 which was selected,amplified and preserved in the glycerol at -70℃.Second,TK gene was liberated from the pRep8.TK (HindⅢ and XbaⅠ) and subcloned into the shuttle plasmid pAdtrack CMV to make pAdtrack CMV TK which was then linearized by PmeⅠand transferred into BJ5183pAdEasy 1 bacteria.Transformants were selected on LB agar plates containing 12 5 μg/mL kanamycin.Plasmid DNA was prepared from individual colonies,amplied and purified.Results The pAdtrack CMV TK(about 10 kb)and pAdEasy CMV TK(about 34 kb),both selected by kanamycin resistance,could be clearly identified by size after fractionating DNA in agarose gel.The simplied two step homologous recombination in bacteria generated recombinant adenovirus plasmids at a frequency of 90%(9/10).Conclusions The simplied two step homologous recombination in bacteria is a convenient and effective way to construct the recombinant adenovirus plasmid which is the most important to construct the recombinant adenovirus. China J Cancer Prev Treat,2003,10(5):473-475

关 键 词：肿瘤 基因治疗 病理学 重组腺病毒 遗传学 质粒 两步法 细菌内同源重组法 

分 类 号：R346[医药卫生—基础医学]