用部分补平法克隆乙型肝炎病毒preS/S基因  

Cloning of preS/S gene of hepatitis B virus by partial filling strategy

作　　者：刘定燮[1] 李德祥 骆抗先[1] 

机构地区：[1]第一军医大学南方医院感染内科,广东广州510515 [2]解放军173医院,广东惠州516001

出　　处：《第一军医大学学报》2001年第1期60-61,共2页Journal of First Military Medical University

摘　　要：目的探讨利用Ｋｌｅｎｏｗ大片段对ＤＮＡ末端作部分补平而使非匹配粘端形成匹配粘端的可行性。方法用 ＢａｍＨＩ、ＸｂａｌＩ消解载体 ｐｃＤＮＡ３，用 ＢｇｌＨ、Ｈｉｎｄ Ⅲ从含乙型肝炎病毒全基因组质粒中切取 ＨＢＶｐｒｅＳ／Ｓ基因片段。 ｐｃＤＮＡ３的 ＸｂａｌＩ及 ｐｒｅＳ／Ｓ基因的 Ｈｉｎｄ Ⅲ切口端经 Ｋｌｅｎｏｗ大片段作部分补平后形成二碱基的互补粘端。连接修饰 后的ｐｒｅＳ／Ｓ基因片段与ｐｃＤＮＡ３并转化后筛选阳性克隆。结果限制性内切酶分析显示ｐｒｅＳ／Ｓ基因被定向克隆于载 体ｐｃＤＮＡ３的预定位点。结论部分补平法是连接非匹配ＤＮＡ粘性末端的较好选择。Objective To examine the feasibility of matching 2 uncomplementary cohesive ends of DNA fragments by partial filling with Klenow fragment. Methods pcDNA-3 was linearized by restriction enzyme BamH I and Xbal I, and HBV preS/S gene fragment was obtained by way of digesting a plasmid containing HBV genome by Bgl Ⅱ and Hind Ⅲ. After partially filled with Klenow fragment via controlling the kinds of dNTP in the reaction buffer, the cohesive ends of Xbal I in pcDNA-3 and that of Hind IIII in preS/S gene were complements to each other between the 2 bases. The modified preS/S gene fragment and pcDNA-3 were subsequently ligated and the product was tranfected into E.coli to select recombinant. Result it was confirmed by restriction analysis that the preS/S gene fragment was inserted into foe pcDNA-3 vector with right direction. Conclusion Partial filling strategy is a good choice when ligating two DNA fragments whose cohesive ends are not complementary.

关 键 词：基因重组 Klenow大片段 乙型肝炎病毒 部分补平法 克隆 preS/S基因 

分 类 号：R373[医药卫生—病原生物学]