以大腸桿菌表現人類表皮生長因子接受體-2之研究

Abstract

人類表皮生長因子接受體基因-2 (human epidermal growth factor receptor, HER-2)又叫neu或c-erbB-2，位於染色體17q12-21.32位置上。其基因產物是個具有1255個胺基酸的穿膜酪胺酸活化□接受體。已有報告指出在人類的乳癌、卵巢癌、胃癌、膀胱癌、腎臟癌、食道癌以及非小細胞肺癌，都可觀察到HER-2基因有放大/過量表現(amplification / overexpression )的情形，而且此HER-2基因放大/過量表現與癌症的轉移及誘導腫瘤細胞產生抗藥性可能有關，因此測定癌細胞HER-2蛋白的表現量有助於做正確的治療及癒後評估。本研究的目的是以重組DNA的方法，在大腸桿菌表現人類HER-2重組蛋白，並用以製造單株抗體，以發展方便的檢驗試劑。我們利用RT-PCR技術，選殖不同長度的HER-2基因包括：(1) ECD(extracellular domain)：核□酸214~2080(胺基酸22~643) (2)ICD(intracellular domain)：核□酸1968~3918(胺基酸607 ~1255)，(3)全段HER-2：核□酸214~3918(胺基酸22~1255)，以聚合酵素連鎖反應增幅之基因構築在pET29b蛋白質表現質體中，並送入E.coli BL21(DE3)內，再利用 IPTG來誘發重組蛋白質表現，實驗發現ECD重組蛋白在誘導後6小時有最大量產生，而ICD及HER-2重組蛋白在誘導後3~6小時有最大量產生。重組蛋白多存於包涵體中，我們先以8M urea將其溶解，再以透析方式完成再折疊，最後用含有Ni2+的樹脂純化出重組蛋白質。 根據免疫酵素分析法及西方墨點法的結果，顯示我們所製造的ECD、HER-2重組蛋白可被多株市售抗體所辨認。故本研究已成功地利用較低成本培養細菌的方法，製造出HER-2重組蛋白質。所表現之兩種蛋白質將用於單株抗體的製造，期望未來可應用於檢驗試劑的開發及其他學術與臨床之研究上。

The proto-oncogene human epidermal growth factor receptor-2 (HER-2), also known as neu or c-erbB-2, is located at chromosome 17q12-21.32 and encodes an 185 kDa transmembrane tyrosine kinase receptor. Gene amplification and/or overexpression of HER-2 was found in several types of human cancers, such as breast, ovarian, gastric, bladder, kidney, esophageal and non-small cell lung carcinoma. This HER-2 overexpression in breast cancer is associated with poor overall survival and might induce metastatic and chemoresistant phenotypes. Therefore, determination of HER-2 expression level in cancer cells may be helpful in therapies and prognosis evalutions. The aim of this study is expressing a recombinant HER-2 protein in Escherichia coli in order to develop a convenient detection kit for diagnostic purpose. We used RT-PCR technology to obtain three human HER-2 gene fragments, including (1) ECD(extracellular domain): nucleotide 214~2080 (related to amino acid 22~643) (2) ICD: nucleotide 1968~3918 (related to amino acid 604 ~1255) (3) HER-2: nucleotide 214~3918 (related to amino acid 22~1255). These gene fragments were ligated into pET29b protein expression vectors and transformed into E.coli BL21 (DE3). The optimal protein expression of ECD was found 6 hours after adding the inducer IPTG, while the optimal expression of ICD and HER-2 was found 3~6 hours after adding IPTG. The recombinant proteins, appearing in inclusion bodies, were dissolved in 8M urea, dialysed in PBS, and purified with Ni-chelating affinity chromatographies. Two of the recombinant proteins, ECD and HER-2, can be recognized by several monoclonal antibodies from commercial resources, suggesting that their amino sequence and / or conformation were similar to human HER-2. These two protein will be used in antibody production and further studies of HER-2 function.