抗诺氟沙星噬菌体单链抗体库的构建与筛选  

Construction and Expression of Recombinant Monoclonal Antibody Against Norfloxacin Using Phage Display Technology

作　　者：郑志明[1,2] 金社胜 肖希龙[2] 候晓林[3] 

机构地区：[1]商务部流通产业促进中心,北京100070 [2]中国农业大学动物医学院兽医药理学系,北京100193 [3]北京农学院动物科技系,北京102206

出　　处：《动物医学进展》2010年第S1期36-41,共6页Progress In Veterinary Medicine

基　　金：国家高技术研究发展计划资助项目(2007AA06Z404)

摘　　要：应用噬菌体展示和重组抗体技术制备抗诺氟沙星(NFLX)单链抗体库,筛选获得抗NFLX高特异性、高亲和力的单链抗体scFv。将NFLX与BSA化学偶联获得的免疫源,免疫Balb/C小鼠,提取脾细胞RNA,反转录获得cDNA。以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的特异性引物,扩增获得VH基因和VL基因。通过SOE-PCR法将VH基因和VL基因通过柔性多肽Linker((Gly4Ser)3)拼接成VH-Linker-VL片段,酶切(sfiⅠ+NotⅠ)处理后,插入噬粒载体pCANTAB5E,并转化宿主菌TG1,通过辅助噬菌体M13K07拯救,构建抗NFLX噬菌体单链抗体库。以包被抗原NFLX-OVA包被96孔板,亲和富集法,经三轮淘筛,间接Elisa初步检测重组scFv的特异性抗原结合活性。成功构建噬菌体单链抗体库,获得鼠源抗NFLX特异性的scFv,噬菌体中scFv插入率为90%,获得库容约为1.3×106的抗NFLX噬菌体单链抗体库,筛选得到5株抗NFLX的scFv。为运用噬菌体展示抗体技术制备特异性抗药物单抗及进一步建立药残检测新技术奠定了基础。To construct a library of mouse single chain variable fragment(scFv) antibody against NFLX using phage display and recombinant antibody technique,and getting the scFv antibody against NFLX with high speciality and affinity by bio-panning.The conjugation of Norfloxacin-bovine serum albumin(BSA)is prepared via carbodiimide method as immunogen.Total RNA was extracted from the spleen cell of the immune Balb/C mouse followed RT-PCR using random primer.The heavy-chain and light-chain variable region genes(VH and VL) repertoire of immunoglobulin were amplified respectively by PCR.Then they were linked by a DNA linker encoding(G1y4Ser)3 as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension) PCR.These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichia coli TGl by electroporation.After rescuing with helper phage M13KO7,a library of phage scFv antibody was got.NFLX-OVA was coated on 96 well plate to perform affinity enrichment panning,and direct Elisa was applied to test the bio-activity against special antigen of the scFv.The recombination rate of the recombinant plasmids scFv-pCANTAB5E is 85%,the titer of the phage antibody library was about 1.3 106 pfu,and 5 strains of mouse scFv antibody phage against NFLX were obtained.The mouse special scFv phage library against NFLX was constructed successfully,and it establishes the foundation for screening the antibiotics residues using the phage display technique.

关 键 词：诺氟沙星 噬菌体展示技术 单链抗体 

分 类 号：R392[医药卫生—免疫学]