人血清白蛋白-白介素-2融合蛋白在CHO细胞中的表达

Expression of Human Serum Albumin-Interleukin-2 Fusion Protein in Chinese Hamster Ovary Cells

DOI：10.3969/j.issn.1673-1689.2017.05.002

中文关键词: 人白介素-2 人血清白蛋白 融合蛋白 中国仓鼠卵巢细胞

英文关键词: human interleukin-2,human serum albumin,fusion protein,Chinese hamster ovary cells

基金项目:

摘要点击次数: 375

全文下载次数: 358

中文摘要:

白细胞介素-2在人体免疫应答中具有重要作用，常用于治疗肿瘤、肝炎和免疫缺陷性疾病等多种疾病，但因其体内半衰期短而限制了其临床应用。文中构建了含有融合蛋白基因HSA-IL-2的pMH3质粒，并通过电转染的方法转入中国仓鼠卵巢细胞（Chinese hamster ovary cell，CHO细胞）中，利用pMH3质粒所携带的neo基因进行筛选，经96孔板培养并筛选得到稳定表达目的蛋白质的重组细胞，融合蛋白HSA-IL-2表达量达到2.26 mg/L。利用反转录PCR和Western blot对重组细胞鉴定后发现，融合蛋白编码基因整合入重组CHO细胞基因组中，而且融合蛋白同时具有HSA和IL-2双重免疫原性。利用对IL-2具有依赖性的CTLL-2细胞进行活性分析后发现，筛选得到的表达细胞分泌得到的融合蛋白具有较高的生物活性，表明成功构建具有表达IL-2生物活性能力的CHO细胞。

英文摘要:

Interleukin-2（IL-2） plays an important role in immune response and is wildly used as therapeutic protein to treat diseases，such as cancer，hepatitis and immunodeficiency. In this study，the pMH3 plasmid containing fusion protein of human serum albumin and IL-2 was constructed and then transfected to Chinese hamster ovary（CHO） cells by electroporation. CHO cells stably expressing the target protein were selected by neo gene in pMH3 plasmid and determined by Dot-blot during 96-plate culture. The titer of HSA-IL-2 was 2.26 mg/L in the 24-plate culture. Reverse transcription PCR and western blot showed that the gene encoding HSA-IL-2 was integrated to genome of recombinant CHO cells and the secreted fusion protein was detected by antibody to be HSA and IL-2. The biological activity of fusion protein was analyzed by IL-2 dependent CTLL-2 cells，and the result showed that the recombinant protein possessed a high activity. In conclusion，the recombinant CHO cells producing biological fusion protein of HSA-IL-2 were successful established.

查看全文 查看/发表评论 下载PDF阅读器