去唾液酸糖蛋白受体H1亚基的原核表达及多克隆抗体的制备

目的在原核系统中表达并纯化去唾液酸糖蛋白受体（ASGPR）H1亚基，制备兔抗人ASGPR1多克隆抗体。方法以质粒pEA1为模板，设计引物，将聚合酶链反应（PCR）扩增产物H1基因克隆到原核表达载体pET-32c中。接种含H1/pET-32c的菌株BL21单菌落至LB肉汤中，1∶100稀释转种后用1 mmol/L终浓度的异丙基硫代-β-半乳糖苷（IPTG）诱导表达，表达产物用Ni^2＋螯合柱亲和纯化，用纯化的ASGPR1免疫新西兰兔制备多克隆抗体。结果H1/pET-32c在原核系统中成功表达和纯化出约50.3 kD大小的融合蛋白，用纯化的H1成功制备了兔抗人H1多克隆抗体，并用6His-H1和GST-H1重组蛋白进行免疫印迹技术（Western blot）分析，证实了抗体的正确性。结论应用多克隆抗体可以检测体内外ASGPR H1亚基基因的表达，为临床上测定血清可溶性ASGPR奠定基础。

Prokaryotic Expression of the H1 Subunit of the Asialoglycoprotein Receptor and the Preparation of Polyclonal Antibody

Objective To express and purify the recombinant protein of the H1 subunit of the asialoglycoprotein receptor,and prepare the rabbit anti-E.coli H1 polyclonal antibody.Methods According to the gene sequence of H1,the primers were designed.The full length cDNA encoding H1 amplified from pEA1 by PCR was subcloned into a prokaryotic expression vector(pET-32c).A single colony of E.coli BL21 containing the plasmid H1/pET-32c was inoculated into LB broth,then diluted to 1/100 into 1 000 ml LB broth and induced with 1 mmol/L IPTG.The recombinant H1 was purified with Ni2 chelating HiTrap HP column.Recombinant H1 was used to immunize the rabbit for preparing polyclonal antibody.Results The recombinant H1 protein about 50.3 kD was expressed in E.coli as inclusion body.rH1 was prepared with Ni2 column purification.The polyclonal antibody was also successfully prepared by H1 recombinant protein and was proved by the methods of Western blot analysis of 6His-H1 and GST-H1 recombinant protein.Conclusion High-level expression of H1 was achieved in E.coli.H1 expression can now be monitored by in vitro and in vivo gene transfer studies with the the polyclonal antibody.The polyclonal antibody can be used to develop a new enzyme-linked immunosorbent assary(ELISA) for the soluble asialoglycoprotein receptor quantification.