中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域的原核表达及复性

Prokaryotic Expression, Purification and Renaturation of CRDH1 and CRDH2 of Chinese Marmata Asialoglycoprotein Receptor

目的对中国旱獭去唾液酸糖蛋白受体(ASGPR)H1和H2亚基糖基识别域(CRD)的克隆、表达、纯化及复性。方法用RTPCR从中国旱獭肝组织中扩增ASGPRCRDH1和CRDH2cDNA,分别将其克隆到原核表达载体pRSETB上,在埃希菌BL21(DE3)pLysS内诱导表达含6个组氨酸标签的融合蛋白。融合蛋白经Ni2+螯合柱亲和纯化后,在体外行透析复性。结果ASGPRCRDH1和CRDH2经原核表达后得到分子量约为22ku和15ku的目的蛋白,以包涵体形式存在。经Ni2+螯合柱亲和纯化后获得纯度大于95%的融合蛋白。利用仅识别天然构像的单克隆抗体对复性后产物进行检测,证明复性成功。结论成功地表达了具有活性的ASGPRCRDH1和CRDH2的融合蛋白,在肝脏疾病的靶向治疗研究中具有潜在的应用价值。

Objective The aim of this study is to construct prokaryotic plasmids expressing carbohydrate recognition domain （CRD） genes--subunit H1 （ CRDH1 ） and subunit H2 （CRDH2） of Chinese parmota asialoglycoprotein receptor （ASGPR）, purify and renature the recombinant proteins CRDH1 and CRDH2. Methods Both CRDH1 and CRDH2 of ASGPR were amplified by RT-PCR from the liver tissues of Chinese marmota and cloned into the prokaryotic vector pRSET-B, respectively. Expression of the 6xHis-tagged recombinant proteins was induced by IPTG in E. coli BL21 （DE3） pLysS. Purification of the recombinant proteins was performed by affinity chromatography with Ni-NTA His-bind resin. The purified recombinant proteins were then renatured in dialysis buffer. Results The recombinant proteins ASGPR CRDH1 and CRDH2 were efficiently expressed as inclusion bodies in prokaryotic cells. The final purified products of DRDH1 and CRDH2 had the molecular weight of 22 ku and 15 ku correspondingly. By using mouse monoclonal antibody against AS- GPR （8D7）, Dot-ELISA proved that both CRDH1 and CRDH2 recombinant proteins were effectively renatured. Conclusion The purified and renatured recombinant proteins ASGPR CRDH1 and CRDH2 presented in this study may be used as targeting molecules for gene therapy of liver diseases.