人脱嘌呤脱嘧啶核酸内切酶hAPE1融合蛋白的构建,纯化及其功能的初步研究.docVIP

人脱嘌呤脱嘧啶核酸内切酶hAPE1融合蛋白的构建、纯化及其功能的初步研究 戴 楠，王 东，李梦侠，曹晓静，曾林立，廖 玲，杨宇馨 （第三军医大学大坪医院野战外科研究所肿瘤中心，重庆 400042） 摘 要：目的　构建人脱嘌呤脱嘧啶核酸内切酶（hAEP1）原核表达载体,在大肠杆菌中表达并纯化融合蛋白，并对其活性进行初步分析。方法　用PCR法扩增hAEP1基因，克隆入pET28a(+)载体中，获得重组质粒pET28a-hAPE1。将重组质粒转化入E.coli BL21(DE3), IPTG诱导表达并鉴定。用His亲和层析技术进行蛋白纯化，得到hAPE1融合蛋白。用Western blot及电泳迁移率变动分析实验分析hAPE1融合蛋白的抗原性及活性。结果　所构建的重组质粒pET28-hAPE1经双酶切及DNA测序鉴定表明构建正确。IPTG诱导表达后，经SDS及Western blot鉴定均在预期位置出现阳性条带，His亲和层析纯化后得到hAPE1融合蛋白经SDS及Western blot鉴定正确。Western blot及电泳迁移率变动分析实验证明hAPE1融合蛋白具有抗原性、AP修复及氧化还原活性。结论　成功构建了人脱嘌呤脱嘧啶核酸内切酶的原核表达载体pET28a-hAPE1，并在E.coli BL21(DE3)中高效表达和纯化得到有抗原性及活性的hAPE1融合蛋白，为进一步进行APE1蛋白的生物学功能研究建立了 关键词：人脱嘌呤脱嘧啶核酸内切酶；原核表达；蛋白纯化；结构与功能 中图分类号： 文献标识码：A Construction, purification and functional characterization of hAPE1 fusion protein DAI Nan, WANG Dong，CAO Xiao-Jing, LI Meng-Xia, ZENG Lin-li, LIAO Ling，YANG Yu-Xin（Cancer Center, Research Institute of Surgery, Daping Hospital，Third Military Medical University , Chongqing 400042，China） Abstract: Objective　To construct a prokaryotic expression vector for human apurinic/apyrimidinic endonuclease (hAPE1) genes，purify its fusion protein and study its activity. Methods　The hAPE1 genes amplified by PCR was cloned into the prokaryotic expression vector pET28a(+). This construction pET28a-hAPE1 was transformed into E.coli BL21 (DE3) and induced to express hAPE1 fusion protein with IPTG. The expressed hAPE1 fusion protein was identified by SDSand Western blot, and purified by affinity chromatographic with His column. Immunogenicity and activity of hAPE1 fusion protein was analyzed by Western blot and EMSA. Results　DNA sequencing, SDSand Western blot showed pET28a-hAPE1 was constructed correctly, and it could express hAPE1 fusion protein well in E.coli BL21 (DE3). The hAPE1 fusion protein, which has immunogenicity, AP repair and redox function, could be purified via affinity chromatographic with His column. Conclusion　In this study, the prokaryotic expression vector of pET28a-hAPE1 vecto