肝型脂肪酸结合蛋白基因的原核表达、纯化和鉴定  

Expression,purification and identification of liver fatty acid binding protein

作　　者：杨海波[1] 柳静[1] 王莲哲[1] 廖春丽[1] 陈兰英[1] 

机构地区：[1]河南城建学院生物工程系,河南平顶山467044

出　　处：《重庆医学》2010年第18期2397-2399,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30470877);河南省重点科技攻关资助项目(092102310006)

摘　　要：目的克隆肝型脂肪酸结合蛋白(LFABP)基因,构建其原核表达载体,并进行表达、纯化及鉴定。方法采用聚合酶链反应(PCR)方法从cDNA中扩增出LFABP基因片段,通过酶切和连接,构建原核表达载体pET-m-LFABP,转化入E.ColiBL21(DE3),经IPTG诱导表达;应用亲和层析、凝胶过滤层析和Lipidex1000凝胶层析分级纯化目的蛋白。圆二色谱分析检测目的蛋白的二级结构和热稳定性。结果 SDS-PAGE分析表明,表达产物的相对分子质量约为15×103,与理论值相一致;应用亲和层析、凝胶过滤层析和Lipidex1000凝胶层析分级纯化目的蛋白,得到纯度较高的蛋白。蛋白的圆二色谱分析表明在大肠杆菌中表达的LFABP蛋白可以形成正确的折叠,热稳定性检测结果表明LFABP蛋白在高温下具有较高的稳定性。结论利用大肠杆菌表达系统,获得了较高纯度的目的蛋白,为进一步研究脂肪酸结合蛋白LFABP的生物学特性、参与脂肪酸代谢调控及其机制研究、与药物的相互作用提供重要基础和研究依据,并将为相关疾病的治疗奠定基础。Objective To construct a prokaryotic expression vector for the expression of LFABP protein,and to express and purify the protein.Methods The LFABP gene was firstly amplified by PCR from genome of cDNA and then cloned into pET-m vector.After sequencing the gene fragment digested by BamHI and XhoI,it expressed in E.Coli DH5α.The expressed protein was identified by SDS-PAGE analysis.To use Ni-NTA purification system,gel filtration chromatography and gel chromatography Lipidex1000 to purify.Results The size of the PCR product was 400 bps and the sequence was identical to the LFABP gene in Genbank.The SDS-PAGE showed that,the relative molecular mass of this protein was consistent with the predicted.The protein was expressed in E.Coli DH5α after the prokaryotic expression vector pET-m-LFABP successfully constructed,and the size of the protein was 15 kd,which was consistent with the theoretical date.The circular dichroism analysis showed that the expressed LFABP protein could form a correct folding in E.coli.The thermal stability analysis showed that the protein had high stability at high temperature.Conclusion The successfully constructed prokaryotic expression vector pET-m-LFABP,and the purified the LFABP protein can not only provide an important foundation to the study of more about the biological characteristics of LFABP,mechanism involved in regulation of fatty acid metabolism and drug interaction,but also provide research proofs to the treatment of related diseases.

关 键 词：肝型脂肪酸结合蛋白 原核表达 纯化 

分 类 号：Q591.5[生物学—生物化学] Q786