HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。

Cloning and Prokaryotic Expression of Extracellular Domain of Human Epidermal Growth Factor Receptor 2

Extracellular domain gene of Human Epidermal Growth Factor Receptor 2(HER2) was amplified by RT-PCR from human breast cancer cell line SK-BR-3.The recombinant expression vector pET30-HER2(Ex) was constructed by inserting the extracellular domain gene of HER2 into the pET-30a vector,and then was transformed into competent E.coli BL21(DE3) cells.After induced by IPTG,the expression of HER2 extracellular domain protein was obtained in E.coli.Proteins from culture medium fraction,periplasmic fraction,soluble cytoplasmic fraction and inclusion body fraction were extracted and analyzed by SDS-PAGE for the localization of HER2 extracellular domain protein.Result of SDS-PAGE revealed that HER2 extracellular domain protein localized in the inclusion body of E.coli cytoplasm.Optimum temperature,IPTG concentration,E.coli cell density(OD600) and induction time were determined for the highest production of HER2 extracellular domain protein.The results showed that when OD600 reached 1.0,induced with 0.1 mmol/L IPTG at 37℃ for 4 h,where the production of recombinant protein was highest.Recombinant protein in the inclusion body of E.coli was extracted with ultrasonic disruption and was purified by Ni2+ affinity chromatography.The obtainment of HER2 extracellular domain protein with high purity and high production lays the foundation for the development of anti-HER2 antibody and the study of HER2 overexpressing tumor vaccine.