人视黄醇结合蛋白4 cDNA全长的克隆、表达和纯化  

Cloning,expression and purification of human RBP4 in E.coli

作　　者：孙玉秀[1] 陈利春 汪凌云[1] 陈兵[1] 陈冠军[1] 鲁云霞[1] 

机构地区：[1]安徽医科大学生化教研室,合肥230032 [2]合肥妇幼保健院,合肥230032

出　　处：《安徽医科大学学报》2010年第3期298-302,共5页Acta Universitatis Medicinalis Anhui

摘　　要：目的构建人视黄醇结合蛋白4(RBP4)cDNA全长的原核表达质粒(pET-28a(+)-hRBP4)并在大肠杆菌中高效表达。方法取成人正常肝脏组织提取总RNA,RT-PCR后的扩增产物回收,构建克隆载体pMD-RBP4,设计带酶切位点BamHⅠ和HindⅢ的引物,PCR后酶切插入pET-28a(+)中,转化BL21(DE3),IPTG诱导表达,Western blot检测其特异表达,并对rhRBP4诱导表达时IPTG的浓度、时间和温度等条件进行了优化;用镍亲和层析柱进行纯化,SDS-PAGE鉴定其纯度。结果测序证实所得的hRBP4 cDNA序列与其在GenBank中的序列基本一致,重组质粒pET-28a(+)-RBP4的双酶切结果与预期大小完全一致,IPTG诱导后高效表达的蛋白质以不溶性包涵体形式存在;Western blot结果证实表达产物为RBP4。IPTG诱导的最佳浓度是0.04mmol/L,时间为6h,温度为37℃,镍亲和层析后经电泳证实其纯度可达96%。结论经诱导表达和纯化的rhRBP4可用于制备抗体和进一步研究。Objective To construct the recombinant plasmid pET-28a（＋）-hRBP4 with human retinol binding protein 4（RBP4）full length cDNA sequence,to express the hRBP4 protein effectively in E.Coli.Methods Total RNA was isolated from normal human liver,then RBP4 were amplified by two-step RT-PCR.The cloning plasmid pMD-hRBP4 was constructed after retrieval of the amplification products,then RBP4 was subcloned with primers containing restriction endonucleases recognition sites of BamHⅠand Hind Ⅲ,ligated into pET-28a（＋）,transducted into BL21（DE3）.After sequenced,the bacteria were induced by IPTG and proteins were expressed,identified with SDS-PAGE and Western blot.Then induction conditions of high level expression were experimented and the fusion protein expressed in E.coli following IPTG induction was purified by Ni2＋ affinity chromatography.The purity of the recombinant protein was detected with SDS-PAGE.Results The sequencing result proved that the cloned hRBP4 cDNA sequence was uniform with its sequence stored in GenBank. The double enzyme cutting result of recombinant plasmid pET-28a（＋）-hRBP4 was coincident with expecting.RBP4 was highly expressed in the form of insoluble inclusion body in E.Coli.Western blot result showed that the highly expressed protein was really RBP4.The best induction concentration of IPTG was 0.04 mmol/L,the best induction time was 6 h and the best induction temperature was 37℃.The result of SDS-PAGE indicated that the purity of rhRBP4 could be reached 96% after Ni2＋ affinity chromatography.Conclusion The induced and pruified protein rhRBP4 could be used to prepare antibody and for future research.

关 键 词：视黄醇结合蛋白质类 

分 类 号：R345.61[医药卫生—基础医学] R392.2