人表皮生长因子受体HER2胞外近膜区基因的克隆及原核表达

目的克隆人表皮生长因子受体HER2胞外近膜区基因,原核表达并纯化重组蛋白。方法从人乳腺癌细胞系SK-Br3中扩增HER2胞外近膜区编码基因,克隆并测序后,插入原核表达质粒pET41d中,转化大肠杆菌BL21(DE3),IPTG诱导表达,并进行纯化。结果从SK-Br3细胞系中扩增出387bp的人HER2胞外近膜区基因;重组表达质粒pET41d/HER2构建正确;IPTG浓度为0.5mmol/L时,目的蛋白的表达量最高;纯化后重组蛋白浓度为1.5mg/ml。结论已成功表达了HER2胞外近膜区蛋白,为抗HER2单克隆抗体的制备提供了抗原。

Cloning and Prokaryotic Expression of Gene Encoding Extracellular Region of Human Epidermal Growth Factor Receptor HER2

Objective To clone the gene encoding extracellular region of human epidermal growth factor receptor HER2 and express in prokaryotic cells. Methods The gene fragment encoding a cyclic domain in extracellular region of HER2 was amplified from human breast cancer cell line SK-Br3, then cloned, sequenced and inserted into prokaryotic expression vector pET41d. The con-structed recombinant plasmid pET41d / HER2 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed protein was purified by affinity chromatography. Results The gene fragment encoding the extracellular region of HER2, at a length of 387 bp, was amplified from SK-Br3 cells, and recombinant plasmid pET41d / HER2 was constructed correctly. The expres-sion level of target protein reached a peak value after induction of recombinant E. coli with 0. 5 mmol / L IPTG. After purification, the concentration of recombinant protein was 1. 5 mg / ml. Conclusion The extracellualr region of HER2 was successfully expressed, which provide an antigen for preparation of McAb against HER2.