| 孟琴 汤伟松 刘亮 李淑珍 唐晓波.人结合珠蛋白cDNA 克隆及其在大肠杆菌中的表达和鉴定[J].,2014,14(24):4644-4647 |
| 人结合珠蛋白cDNA 克隆及其在大肠杆菌中的表达和鉴定 |
| Cloning, Expression and Identification of Human Hp cDNAin Escherichia coli |
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| DOI： |
| 中文关键词: 卵巢癌 人结合珠蛋白 克隆 表达 纯化 |
| 英文关键词: Oophoromas Human haptoglobin Cloning Expression Identification |
| 基金项目:哈尔滨市科技创新人才研究专项资金项目（2008RFXXS018） |
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| 中文摘要: |
| 目的：克隆人结合珠蛋白（haptoglobin,Hp）cDNA ，并在大肠杆菌中表达和鉴定。方法：从Hela 细胞中分离总RNA，采用RT-PCR 方法获得人Hp cDNA，分别克隆至原核表达载体pET-32a和PGEX-4T-1,转化至大肠杆菌BL21，IPTG 诱导表达，并进行SDS-PAGE 及Western blot 鉴定。结果: 成功构建了高效原核表达质粒PET-32a-Hp 和PGEX-4T1-Hp;Western 印迹结果表明,经IPTG 诱导,在大肠杆菌中表达了分子量约30 kD和37 kD 的目的蛋白；表达产物经Ni2+-NTA 离子交换树脂纯化, 纯度>90%。结
论：在E.coli成功表达和纯化了人Hp 融合蛋白，为进一步开发人Hp 诊断试剂打下基础。 |
| 英文摘要: |
| Objective:To clone, express and identificate human Haptoglobin cDNA in Escherichia coli(E.coli).Methods:Human
Hp cDNA was obtained from total RNA isolated from Hela cells by reverse transcription PCR method, then it was inserted into
prokaryotic expression vectors pET-32a and PGEX-4T-1 for the IPTG-induced expression in E.coli BL21, and it was identified by
SDS-PAGE and Western blot.Results:The expression plasmid pET-32a-Hp and PGEX-4T-1-Hp were constructed successfully. Western
blot analysis showed that fusion protein pET-32a-Hp with 30kD molecular weight and PGEX-4T-1-Hp with 37kD expressed in E.coli.
The purification of fusion protein was more than 90%after purification using Ni2+-NTA ion exchange resin.Conclusion:Fusion protein
human Hp was successfully expressed in E.coli and purified, which settled a foundation for further development of human Hp diagnostic
reagents. |
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