新型噬菌粒展示载体pCANTAB5L的构建  

Construction of a novel phagemid pCANTAB5L

作　　者：沈毅珺[1] 潘卫[1] 易进华 徐容[3] 陈秋莉[1] 潘欣[1] 冯春芝[1] 邓松华[3] 戚中田[1] 刘彦君 

机构地区：[1]第二军医大学基础医学部微生物学教研室,上海200433 [2]上海复旦张江生物医药股份有限公司 [3]安徽医科大学病理生理学教研室

出　　处：《第二军医大学学报》2003年第3期298-302,共5页Academic Journal of Second Military Medical University

基　　金：国家"8 6 3"计划项目 (2 0 0 1AA2 15 0 5 1;2 0 0 2AA2Z330 9)

摘　　要：目的 :将柔性多肽接头和多克隆位点引入噬菌粒载体 pCANTAB5X中 ,构建适用于展示各种随机肽库、功能靶蛋白、功能靶蛋白变异体库和大分子量 (>30 0个氨基酸 )功能蛋白的新型噬菌粒展示载体。 方法 :应用 5′端含XbaⅠ、StuⅠ、SalⅠ、KpnⅠ识别序列的引物 ,PCR扩增获得XbaⅠ StuⅠ SalⅠ KpnⅠ [G4S]3 NotⅠ衔接片段 ,将该PCR产物克隆到pMD 18T中 ,再将该片段插入 pCANTAB 5X中构建成新型噬菌粒展示载体 pCANTAB5L。 结果 :限制性内切酶酶谱及DNA序列分析证明[G4S]3多肽接头和限制性内酶切位点XbaⅠ、StuⅠ、SalⅠ、KpnⅠ序列被引入到新构建的噬菌粒载体pCANTAB 5L中。 结论 :成功构建了新型噬菌粒展示载体 pCANTAB5L ,并能有效展示功能靶蛋白。Objective: To construct the new phage display vector for effective display of the random peptide library, functional protein, functional protein mutant library and large protein (>300 amino acids), the flexible peptide linker and the multiple clone sites were introduced into the phagemid pCANTAB5X. Methods: The linker fragment Xba Ⅰ Stu Ⅰ Sal Ⅰ Kpn Ⅰ 3 Not Ⅰ was generated by PCR amplification with the upstream primer containing Xba Ⅰ, Stu Ⅰ, Sal Ⅰ and Kpn Ⅰ recognition sequence and the downstream primer. The PCR product was inserted into pMD 18T and then cloned into pCANTAB5X to construct the phagemid pCANTAB5L. Results: A novel phagemid pCANTAB5L was successfully constructed with 3 peptide linker and new cloning sites including Xba Ⅰ, Stu Ⅰ, Sal Ⅰ, Kpn Ⅰ. Conclusion: The novel phagemid can effectively display the functional protein, functional protein mutant library and large protein.

关 键 词：噬菌体展示 噬菌粒载体 多克隆位点 多肽接头 

分 类 号：Q782[生物学—分子生物学]