可分泌表达Aβ阻断肽融合基因原核表达载体的构建  

Construction of expression vector for ABAD-DP fusion gene

作　　者：杨宇[1] 杨欣[2] 孙欣[1] 吴昊[3] 吴江[1] 

机构地区：[1]吉林大学第一医院神经内科,吉林长春130021 [2]上海交通大学瑞金医院神经内科 [3]吉林大学第一医院肾病科,吉林长春130021

出　　处：《中国老年学杂志》2010年第18期2609-2612,共4页Chinese Journal of Gerontology

基　　金：国家自然科学基金资助课题(30872721);国家自然科学青年基金资助课题(30801211);高等学校博士学科点专项科研基金新教师基金资助课题(200801831073);吉林大学研究生创新研究计划(20101034)

摘　　要：目的构建神经营养素3(NT3)信号肽、Aβ1-42阻断肽ABAD-DP、溶源肽HA2及穿膜肽TAT融合基因的原核表达载体pBV220/NAHT,为将Aβ1-42阻断肽ABAD-DP用于AD基因治疗提供实验基础。方法采用非对称互补引物/模板法,分别制备两端含有酶切位点的ABAD-DP和HA2-TAT的cDNA,将其连接到NT3的信号肽3′端,组成融合基因NT3-ABAD-DP-HA2-TAT(NAHT),再将该融合基因亚克隆于原核表达载体pBV220,构建原核表达载体pBV220/NAHT。结果经DNA测序证实成功克隆ABAD-DP和HA2-TATcDNA;经限制性内切酶酶切证实成功将ABAD-DP和HA2-TAT基因重组到NT3信号肽的3′端,并将融合基因亚克隆于pBV220载体内。结论成功构建了表达NT3信号肽、阻断肽ABAD-DP、融源肽HA2及穿膜肽TAT融合基因的原核表达载体pBV220/NAHT。Objective To construct the prokaryotic expression vector bearing fusion gene NT3-ABAD-DP-HA2-TAT and lay a foundation for the further study on genetic therapy of AD.Methods By means of asymmetrical primer/template,double stranded cDNA of ABAD-DP and HA2-TAT were gained respectively,which included restriction enzymes sites on the extremities.Then they were ligated to the signal and leader peptides of neurotrophin 4,the fusion gene was named NAHT.Then it was subcloned into prokaryotic expression vector pBV220,called pBV220/NAHT.Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that ABAD-DP and HA2-TAT cDNA to the 3＇ terminal of the signal peptides of NT3 were recombined,and the fusion gene was subcloned into pBV220 successfully.Conclusions Prokaryotic expression vector pBV220/NAHT can be constructed successfully.

关 键 词：神经营养素3 ABAD-DP 基因治疗 原核表达载体 

分 类 号：Q78[生物学—分子生物学]