摘要
目的 :利用大肠杆菌高效表达肝癌相关抗原HAb18G胞外区片段 ,并分析其与相应抗体的结合活性 ,进而明确表达产物用于筛选基因工程抗体的可行性 .方法 :用PCR技术扩增出HAb18G胞外区基因片段 ,克隆入原核表达载体pGEX 4T 3中 ,筛选及鉴定阳性重组子 ,IPTGv诱导表达后对表达产物进行纯化及复性等处理 ,同时利用SDS PAGE ,Western Blot以及ELISA等方法检测蛋白表达情况及其免疫学特性 .结果 :成功构建了肝癌相关抗原HAb18G胞外区片段的原核表达载体 pGEX 4T 3/HAb18GE ,并在大肠杆菌中成功实现高效融合表达 .表达蛋白Mr 4 6 0 0 0 ,表达量约占菌体总蛋白量的 4 3% ,表达产物主要以包涵体形式为主 .另外 ,表达蛋白在变性和非变性的条件下均可以与HAb18GmAb特异性结合 .结论 :原核融合表达的HAb18G胞外区片段可以替代天然的HAb18G蛋白 ,以用做抗原来筛选新型的基因工程抗体 .
AIM: To efficiently express the extracellular domain of hepatoma associated antigen HAb18G in E.coli and check the immunologic characteristics of expressed protein. Furthermore, to assure whether it can be used as antigen in selecting specific gene engineering antibody. METHODS: Extracellular domain of HAb18G was amplified by PCR. After restriction endonuclease digestion, it was inserted into a prokaryotic expression vector pGEX 4T 3. Positive E.coli clones containing recombinant plasmid pGEX 4T 3/HAb18GE were selected and identified. After induction with IPTG, the expressed fusion protein was purified and renatured. Expression was checked by SDS PAGE and Western Blot. The immunologic characteristics of expressed protein was analyzed through ELISA under the condition of denaturation and renaturation. RESULTS: We successfully constructed a recombinant prokaryotic expression vector pGEX 4T 3/HAb18GE and efficiently expressed extracellular domain of HAb18G in E.coli . The relative molecular mass ( M r) of expressed protein was about 46 000 and its amount almost held 43% of total bacterial proteins and the protein was presented mainly in the form of inclusion body. In addition, the protein could specially bind to McAb of HAb18G under the condition of denaturation and renaturation. CONCLUSION: The extracellular domain of HAb18G expressed in prokaryotic expression system can substitute natural HAb18G protein as antigen to screen new type gene engineering antibody of HAb18G.
出处
《第四军医大学学报》
北大核心
2003年第12期1057-1060,共4页
Journal of the Fourth Military Medical University
基金
国家 8 63计划重点课题 (2 0 0 1AA2 1 51 0 1 )
关键词
肝肿瘤
抗原
肿瘤
胞外区
融合表达
liver neoplasms
antigens, neoplasms
extracellular domain
fusion expression
