截短型BAP31/GST基因重组质粒的构建及融合蛋白的表达、纯化和鉴定  

Prokaryotic expression,purification and identification of truncated BAP31/GST fusion protein

作　　者：李海涛[1] 宋朝君[1] 孙元杰[1] 魏玉英[1] 李永明[1] 刘飞[1] 刘志佳[1] 张葵[1] 金伯泉[1] 杨琨[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,西安710032

出　　处：《免疫学杂志》2011年第1期1-4,共4页Immunological Journal

基　　金：国家高技术研究发展计划(863计划)重大项目(2006AA02A237);国家自然科学基金面上项目(30872371)

摘　　要：目的构建编码截短型肿瘤抗原BAP31(△BAP31)与GST融合基因的原核表达载体,在大肠杆菌中表达并对融合蛋白(△BAP31/GST)进行纯化和初步鉴定。方法 PCR扩增编码△BAP31的基因片段,上下游分别引入EcoR I及Xho I酶切位点,亚克隆至含有GST标签的原核表达载体pGEX4T-1中,构建重组表达载体pGEX4T1-△BAP31,将该载体转化大肠杆菌DH5α,IPTG诱导表达△BAP31/GST,用GST亲和层析分离纯化原核表达的△BAP31/GST,表达产物分别用SDS-PAGE和West-ern blot进行鉴定。结果重组质粒经限制性内切酶EcoR I和Xho I双酶切鉴定和IPTG诱导表达△BAP31/GST的SDS-PAGE分析表明,表达产物的相对分子质量为40 000,与理论值相符,并主要以可溶性蛋白形式存在;经过对融合蛋白表达条件的优化,在IPTG浓度为1 mmol/L,诱导6 h目的蛋白表达量最高;灰度扫描分析发现,融合蛋白表达量占菌体蛋白总量的70.6%,纯化产物的纯度最高可达94.5%,Western blot证实该△BAP31/GST可与抗GST单克隆抗体(mAb)发生特异性结合反应,分子量为△BAP31与GST分子量之和,提示为融合蛋白。结论成功构建了编码△BAP31基因原核表达载体pGEX4T1-△BAP31,利用大肠杆菌表达系统和GST亲和层析,获得了较高纯度的△BAP31/GST融合蛋白,为进一步研究肿瘤抗原BAP31的功能及开发以BAP31作为靶点的肿瘤疫苗提供了试验基础。In this study,we aimed to construct a expression plasmid for△BAP31 gene and identify the recombinant protein expression.Firstly,the△BAP31 coding sequence was amplified by polymerase chain reaction（PCR） method and subcloned into pGEX-4T1vector.After the target gene was sequenced,the plasmid was transformed into E coil DH5α and induced with IPTG to express fusion protein △BAP31/GST which was then proved by SDS-PAGE and Western blot.SDS-PAGE analysis showed that a novel protein with the expected molecular mass about 40 000 was expressed with the inducement of IPTG.The fusion protein was existed mostly in the form of soluble protein,and grayscale scanning showed that the expressed △BAP31/GST fusion protein was accounted for 70.6% of the total bacterium protein.The purity of the fusion protein reached 94.5% after purification by High-Affinity GST Resin.Then western blot confirmed the recombinant protein was △BAP31/GST fusion protein.In our study,high purification △BAP31/GST fusion protein is obtained through the E.coil expression system.

关 键 词：截短型BAP31(△BAP31) GST 融合蛋白 蛋白表达 纯化 

分 类 号：Q78[生物学—分子生物学]