抗HBs Fab-IFNα融合蛋白的制备与初步鉴定  

Preparation and preliminary identification of anti HBs Fab-IFNα fusion protein

作　　者：陆慧琦[1] 宋杰[1] 叶伟民[1] 韩焕兴[1] 

机构地区：[1]第二军医大学长征医院实验诊断科,上海200003

出　　处：《解放军医学杂志》2011年第5期467-470,共4页Medical Journal of Chinese People's Liberation Army

基　　金：上海市科委资助项目(074119642)

摘　　要：目的构建抗HBsAg的pComb Fab-IFNα载体,原核表达具双重生物活性的抗HBs Fab-IFNα融合蛋白。方法以pBAD-Fab和pBADIFNα作为模板,分别扩增Fd、λ和IFNα基因,经相应的限制性内切酶酶切后分3次克隆入pComBHss质粒,转化XL-1 Blue大肠埃希菌。限制性酶切和测序鉴定重组质粒,免疫蛋白印迹（Western blotting）和斑点印迹（Dot blotting）鉴定融合蛋白的表达及抗原结合活性。结果重组载体的酶切、电泳及测序表明抗HBs Fab-IFNα基因克隆正确。表达产物经12%SDS-PAGE电泳、转印,Western blotting显示该融合蛋白分子量约为65kD,Dot blotting显示其与HBsAg具有结合能力。细胞病变抑制法测定IFNα生物学活性为7.8×1045.1×105U/ml。结论该原核系统成功表达了抗HBs Fab-IFNα融合蛋白,表明其既具有抗HBsAg结合能力,又具备IFNα的生物活性,为进一步的系统表达和应用研究提供了条件。Objective To construct the anti HBsAg pComb Fab-IFNα vector,and to express the fusion protein consisting of IFN and anti-HBs Fab in prokaryon.Methods Using pBAD-IFN plasmid and pBAD-Fab plasmid as template,the anti-HBs Fd,and IFN were amplified separately with corresponding endonuclease sites by polymerase chain reaction（PCR）.Each PCR product was digested with specific endonuclease and inserted into pComBHss vector,and then transformed into XL-1 Blue.The recombinant plasmid was isolated by miniprep for restriction analysis and sequencing.Fusion protein was identified by Western blotting and Dot blotting.Results The recombinant plasmid was confirmed by restriction electrophoresis and sequencing.Aliquots of human anti-HBs Fab-IFNα were concentrated and size fractionated by 12% SDS-PAGE and stained with Coomassie.The proteins were transferred to nitrocellulose,incubated with HRP-conjugated goat anti-human IgG Fab and Rab anti-human IFN.The molecular weight of the fusion protein was about 65kD.Anti HBs Fab-IFNα fusion protein showed satisfactory bioactivity by Western blotting,and HBsAg affinity was confirmed by Dot blotting.The IFN activity was 7.8×104-5.1×105U/ml.Conclusions The recombinant expression plasmid pComb Fab-IFNα has been constructed successfully in E.coli.The successful expression of the fusion protein with both HBsAg affinity and Fab-IFNα activity paves a way of targetins hepatitis B virus in the treatment of hepatitis.

关 键 词：免疫球蛋白Fab碎片 干扰素Α 肝炎表面抗原 乙型 肝炎病毒 乙型 

分 类 号：R392.11[医药卫生—免疫学]