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Prokaryotic expression and immunological analysis of the extracellular domain of hepatoma associated antigen HAb18G
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 12
- DOI:
- 10.3321/j.issn:1000-2790.2003.12.001
- Key Word:
- 肝肿瘤;抗原,肿瘤;胞外区;融合表达
Abstract： 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 46 000,表达量约占菌体总蛋白量的43%,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HAb18G mAb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.
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