具有细胞穿透功能的串联亲和层析分离载体的构建与功能鉴定  

Construction and functional identification of tandem affinity purification vector with transmembrane function

作　　者：邓鹏[1] 刘芸[1] 李涛 吴向玲[1] 刘亚伟[1] 姜勇[1] 

机构地区：[1]南方医科大学基础医学院病理生理学教研室,教育部重大疾病转录组与蛋白质组学重点实验室,广州510515

出　　处：《解放军医学杂志》2010年第5期547-550,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金面上项目(30572151);教育部长江学者和创新团队发展计划(IRT0731);国家自然科学基金委员会-广东省人民政府自然科学联合基金(U0632004);广州市科技计划(2007J1-C0301)

摘　　要：目的建立基于抗生蛋白链菌素结合肽(SBP)-钙调蛋白结合肽(CBP)分离标签以及细胞穿透肽转录反式激活因子(TAT)和绿色荧光蛋白(GFP)原核表达系统的、具有细胞穿透功能的原核串联亲和层析分离系统。方法应用PCR方法,以pEG-FP-C2载体为模板扩增EGFP序列,以pNTAP载体为模板扩增CBP-SBP序列,采用退火方法得到TAT片段,采用常规酶切、连接方法将EGFP、CBP-SBP和TAT片段依次克隆入pET14b-MCStop载体中,酶切、PCR和测序鉴定无误后,得到pET14b-CBP-SBP-EGFP-TAT重组质粒。将重组质粒转化BL21(DE3)大肠埃希菌,以IPTG诱导融合蛋白表达,利用Ni2+-NTA亲和层析纯化获得融合蛋白,将不同浓度的融合蛋白加入培养的人肝癌细胞株(HepG2)检测融合蛋白的穿细胞膜功能。结果所构建的pET14b-MCS-CBP-SBP-EGFP-TAT融合蛋白表达载体正确,该载体可在大肠埃希菌内高效表达,用Ni2+-NTA纯化获得了相对分子量约40kD的目的融合蛋白。细胞功能实验结果表明融合蛋白能够穿越细胞膜,且具有浓度依赖性。结论成功构建了pET14b-MCS-CBP-SBP-EGFP-TAT融合蛋白表达载体,并获得了具有良好穿透真核细胞膜功能的融合蛋白,为进一步研究细胞内蛋白相互作用、细胞穿透肽TAT的功能及其穿透细胞的机制奠定了基础。Objective To construct tandem affinity purification vector with calmodulin-binding peptide （CBP）, streptavidin-binding peptide （SBP）, trans-activator of transcription （TAT） and green fluorescent protein （GFP）, and identify its transmembrane function. Methods EGFP and CBP-SBP sequences were amplified respectively from pEGFP-C2 and pNTAP vectors by PCR, and TAT fragment was obtained by annealing, and then subcloned into pET14b-MCStop by routine enzyme digestion and connection. The double-stranded DNA of TAT obtained with annealing method was then further subcloned into pET14b-CBP-SBP-EGFP to obtain prokaryotic expression vector pET14b-MCS-CBP-SBP-EGFP-TAT. The vectors were confirmed by enzyme digestion, PCR and DNA sequencing, and then transformed into BL21 （DE3） competent cells. pET14b-MCS-CBP-SBP-EGFP-TAT fusion proteins were induced with IPTG and then purified by Ni2＋-NTA affinity chromatography. Different concentrations of the purified protein were added into cultured human liver carcinoma cell strain （HepG2）, and the fluorescent proteins in cells were detected with fluorescent microscopy. Results The prokaryotic expression vectors of pET14b-MCS-CBP-SBP-EGFP-TAT were successfully constructed, and the fusion protein was highly expressed in E. coli. The high-purified fusion protein （His-CBP-SBP-EGFP-TAT） with relative molecular mass of approximately 40kD was obtained by Ni2＋-NTA affinity chromatography, which could penetrate the cell membrane in a concentration dependent manner. Conclusions Tandem affinity purification vector of pET14b-MCS-CBP-SBP-EGFP-TAT has been successfully constructed, and the purified fusion protein with transmembrane function has been obtained, which provides a tool for future study of transmembrane mechanism of TAT and protein-protein interactions in cells.

关 键 词：基因产物 tat 色谱法 亲和 抗生蛋白链菌素 钙调蛋白结合蛋白质类 

分 类 号：R373.2[医药卫生—病原生物学] R392.32[医药卫生—基础医学]