抗磺胺二甲氧嘧啶VHH抗体噬菌体库的构建和鉴定  

Construction and identification of a phage library of anti-sulfadimethoxine VHH antibody

作　　者：贺东阳 张齐 张星星[2] 钟发刚[2] 张小莺[1,2,3] 王金泉 HE Dongyang;ZHANG Qi;ZHANG Xingxing;ZHONG Fagang;ZHANG Xiaoying;WANG Jinquan(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production,Xinjiang Academy of Agricultural and Reclamation Sciences,Shihezi 832000,China;Department of Biomedical Sciences,Ontario Veterinary College,University of Guelph,Ontario N1G2W1,Canada)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000 [3]Department of Biomedical Sciences,Ontario Veterinary College,University of Guelph,Ontario N1G2W1,Canada

出　　处：《畜牧与兽医》2021年第1期48-53,共6页Animal Husbandry & Veterinary Medicine

基　　金：乌鲁木齐市科技计划(P16130001)。

摘　　要：旨在制备抗磺胺二甲氧嘧啶(SDM)驼源单域重链(VHH)抗体,用于检测动物源性食品中SDM的残留。采用重氮化法,SDM分别与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联,合成人工免疫原(SDM-BSA)和包被抗原(SDM-OVA)。用SDM-BSA免疫骆驼,在第5次免疫后1周采集骆驼外周血液,分离外周血淋巴细胞,提取RNA,RT-PCR扩增VHH基因,将VHH基因插入pCANTAB-5E噬菌粒载体,构建双峰驼单域重链抗体库。经本实验室前期测定其库容为1.08×10^(5) CFU,阳性率为96.6%。利用噬菌体展示技术经过4轮生物淘选,筛选获得特异性表达抗SDM抗体的重组噬菌体。结果:构建了SDM的驼源VHH抗体库,经生物淘选及phage ELISA检测获得了特异性的重组噬菌体。通过4轮生物淘选,获得了能够与SDM-OVA抗原有明显结合力且能够特异性表达抗SDM抗体的重组噬菌体,为后期检测SDM在动物源性食品残留奠定了良好的基础。To prepare a camel-derived variable domain of heavy-chain antibody(VHH)against sulfadimethoxine(SDM)for the detection of SDM residues in animal foods.Diazotization was used to couple SDM with bovine serum albumin(BSA)and chicken ovalbumin(OVA)to synthesize artificial immunogen SDM-BSA and coated antigen SDM-OVA.Experimental camels were immunized with SDM-BSA,and peripheral blood samples were collected from the camels one week after the fifth immunization,peripheral blood lymphocytes were isolated,RNA was extracted,and the VHH gene was amplified by RT-PCR.The VHH gene was inserted into the pCANTAB-5 E phage vector to construct a bactrian camel heavy chain antibody library with a laboratory pre-determined storage capacity of 1.08×10^(5) CFU and the positive rate was 96.6%.After four rounds of biological panning using the phage display technology,specific recombinant phages were screened.As a result,a camel-derived anti-SDM VHH antibody library was constructed specific recombinant phages were obtained by bio-panning and phage ELISA detection.The recombinant phage that could bind to the SDM-OVA antigen and specifically express anti SDM antibody was obtained through four rounds of biological panning,which laid a good foundation for the subsequent experiments.

关 键 词：磺胺二甲氧嘧啶(SDM) 单域重链抗体(VHH) 噬菌体展示技术 

分 类 号：S852.43[农业科学—基础兽医学]