新基因MAGE-n表位肽与人HSP70融合基因的构建及表达纯化  

Construction,expression and purification of a fusion gene of MAGE-n peptide with human heat shock protein 70

作　　者：张秀敏[1] 史琪琪[1] 李增山[1] 叶菁[1] 王军伟[1] 林慧[1] 曲萍[2] 胡沛臻[1] 隋延仿[1] 

机构地区：[1]第四军医大学基础部病理与病理生理学教研室/肿瘤生物学国家重点实验室,西安710032 [2]第四军医大学教学实验中心

出　　处：《中国医药生物技术》2010年第4期257-260,共4页Chinese Medicinal Biotechnology

基　　金：国家高技术研究发展计划(863计划)(2007AA02Z470);国家自然科学基金(30901735);陕西省攻关项目[2007K09-05(2)]

摘　　要：目的构建MAGE-n159-167(QLVFGIEVV)表位肽与人热休克蛋白70(HSP70)的融合基因并表达纯化。方法应用PCR方法,将QLVFGIEVV的cDNA序列融合到人HSP70基因的3'端,将融合基因克隆入原核表达载体pET-28a(+)中,构建重组质粒pET-28a(+)QLVFGIEVV-HSP70;采用双酶切(Sac Ⅰ、Hind Ⅲ)及PCR鉴定后测序;转化大肠杆菌E.coli BL21(DE3),用IPTG诱导表达,Ni Sepharose6FF亲和填料进行分离纯化,SDS-PAGE检测表达及纯化结果。结果经PCR扩增成功获得约2.0kb的目的片段,重组体经Sac Ⅰ、Hind Ⅲ双酶切分析及PCR结果与预期结果一致,测序正确。转入重组质粒的E.coli BL21(DE3)经IPTG诱导、SDS-PAGE分析得到71kD左右的目的蛋白条带。用Ni Sepharose6FF亲和填料分离纯化,获得了纯化的融合蛋白。结论成功构建原核表达载体pET-28a(+)QLVFGIEVV-HSP70,并获得纯化的融合蛋白,为基于MAGE-n的肿瘤疫苗研制提供良好的抗原奠定了实验基础。Objective To construct and express a fusion gene of MAGE-n peptide （QLVFGIEVV） with human heat shock protein 70 （HSP70）,and then purify the fusion protein. Methods A cDNA fragment encoding QLVFGIEVV was added to 3 terminus of human HSP70 gene by PCR amplification. The PCR products of fusion gene were cloned into pET28a （＋） vector. The recombinant plasmid pET28a （＋）/QLVFGIEVV-HSP70 was identified by enzyme digestion analysis and sequencing,the plasmids were transformed into E.coli BL21 and were efficiently expressed after IPTG induction. The fusion protein was purified with Ni Sepharose 6FF affinity chromatography. Results Conclusion cloned gene was 2.0 kb in length. Enzyme dissection analysis,PCR and DNA seguencing showed that the products was the same as the expected gene. SDS-PAGE showed that a Mr 71 000 fusion protein was expressed. The expression protein was purified with Ni Sepharose 6FF affinity chromatography,and the purified target fusion protein was obtained. Conclusions Prokaryotic expression vectors pET28a（＋）/QLVFGIEVV-HSP70 and the purified fusion proteins were successfully obtained. The successful expression and purification of the fusion protein has laid the foundation for using it to study tumor vaccine of MAGE-n.

关 键 词：HSP70热休克蛋白类 基因融合 癌症疫苗 MAGE-N 表位肽 原核表达 

分 类 号：R392[医药卫生—免疫学]