摘要
目的构建树鼩载脂蛋白AV原核表达载体并利用His标签纯化该蛋白。方法从树鼩肝细胞中提取总RNA,经逆转录合成cDNA第一链,以cDNA第一链为模板,扩增载脂蛋白AV基因。PCR扩增产物和pET32a原核表达载体经过双酶切,将纯化回收的酶切产物按适当的摩尔比通过T4 DNA连接酶16℃连接过夜,连接产物转化大肠杆菌感受态细胞Top10,挑克隆测序。将克隆成功的载脂蛋白AV重组质粒转化大肠杆菌表达菌株BL21(DE3),利用异丙基-β-D-硫代半乳糖苷诱导表达,并优化诱导条件。表达的载脂蛋白AV经镍离子螯合树脂纯化,采用BCA法分析蛋白浓度,纯度测定采用聚丙烯酰胺凝胶电泳结合薄膜凝胶扫描分析获得。结果挑选的克隆经测序证实载脂蛋白AV基因已经成功连接入pET32a原核表达载体中。在大肠杆菌BL21(DE3)中经异丙基-β-D-硫代半乳糖苷诱导表达,有分子量大小约60 kDa的特异性蛋白产生,与预期大小一致。重组蛋白诱导的最佳表达时间为5 h左右,最佳浓度约在20μmol/L。镍离子螯合树脂柱亲和层析获得的纯化蛋白,经过聚丙烯酰胺凝胶电泳后薄膜凝胶扫描分析显示目的蛋白的纯度在95%以上。结论成功构建了载脂蛋白AV的原核表达载体,该蛋白在大肠杆菌BL21(DE3)中实现了高效表达,纯化后的载脂蛋白AV纯度约95%,为进一步研究其结构与功能奠定了基础。
Aim To construct recombinant plasmid pET32a-apolipoprotein AV and purify the protein with His tag. Methods The total RNA was extracted from tree shrew liver tissues, cDNA was then obtained by reverse tran- scription-polymerase chain reaction. Apolipoprotein AV gene fragment was amplified by PCR. The amplified products and pET32a plasmid were digested by restriction enzymes Xho I and Eco RI, and then the purified products were ligated by T4 DNA ligase. The reeombinants were transformed into E. coli Topl0 and BL21 （ DE3 ）. The apolipoprotein AV was in- duced with isopropy-13-D-thiogalactoside. The expressed conditions were optimized and purified by Nickel ion chelating resin. Purity analysis of the apolipoprotein AV was obtained by SDS-PAGE. Results Recombinant plasmid pET32a- apolipoprotein AV was successfully constructed and expressed in BL21 （ DE3 ）. Isopropy-13-D-thiogalactoside-induced tar- get protein （ about 60 kDa） was detected. The purity of recombinant tree shrew apolipoprotein AV was greater than 95%. Conclusions Recombinant plasmid is successfully constructed, with efficient expression of apolipoprotein AV in E. coli BL21 （DE3） , and has laid down the foundation for further investigation on its structure and functions.
出处
《中国动脉硬化杂志》
CAS
CSCD
北大核心
2013年第9期775-779,共5页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(81270948
81070634
81170381)
北京医院青年启动基金(BJ-2011-30)资助
关键词
树鼩
载脂蛋白AV
原核表达
蛋白纯化
His标签
Tree Shrew
Apolipoprotein AV
Prokaryotic Expression
Protein Purification
His Tag
