抗克百威单链抗体的可溶性表达、纯化与鉴定  

Soluble expression,purification,and identification of scFv against carbofuran

作　　者：詹屋强 黄宇婷[1] 张瑾如 李家冬[1] 刘姚 沈兴 王弘[1] 

机构地区：[1]华南农业大学食品学院广东省食品质量与安全重点实验室畜禽产品精准加工与安全控制技术国家地方联合工程研究中心(广东),广东广州510642

出　　处：《食品工业科技》2017年第20期77-82,89,共7页Science and Technology of Food Industry

基　　金：国家重点研发计划项目(2016YFE0106000);国家自然科学基金(31271866);广东省自然科学基金(2014A030311043);广东省科技计划项目(2014A050503059);广州市科技计划项目(2014J4100185)

摘　　要：在前期获得了抗克百威单链抗体基因的基础上,为进一步研究克百威单链抗体的性质,本文构建了两种抗克百威单链抗体可溶表达载体,p ET22b(+)-C-6His-CBF-sc Fv和p ET22b(+)-N-6His-CBF-sc Fv。比较His标签处于不同位置时,目的蛋白与Ni结合作用,发现His标签处于N端的目的蛋白与Ni结合作用比His标签处于C端时要强。对影响N-6His-CBF-sc Fv可溶性表达的条件进行优化,进而对其进行纯化。结果表明,当选用LB培养基,表达温度18℃,表达时间为16 h,IPTG浓度为1 mmol/L时可溶表达效果最好,可溶表达的目的蛋白量占总目的蛋白量从优化前的10%增加到50%。优化表达后的N-6His-CBF-sc Fv破壁上清经Ni Sepharose HP金属螯合亲和层析及阳离子交换层析两步纯化,纯化后纯度达90%。经ic ELISA鉴定,抗克百威单链抗体保持抗原结合活性,IC_(50)值为63.80 ng/m L。获得的抗克百威单链抗体可用于后续特性研究。The lab constructed anti-carbofuran sc Fv gene,for further study of the nature of anti-carbofuran sc Fv,two kinds of anti-carbofuran sc Fv soluble expression vector p ET22b（ ＋）-C-6His-CBF-sc Fv and p ET22b（ ＋）-N-6His-CBF-sc Fv were constructed.N-6His-CBF-sc Fv was better than C-6His-CBF-sc Fv for separation. The conditions of soluble expression and purification effect factors about N-6His-CBF-sc Fv were optimized.The results showed that soluble expression was the best when LB medium was used,the expression temperature was 18 ℃,the expression time was about 16 hours and the concentration of IPTG was 1 mmol/L.Ni Sepharose HP and Iex Cap SP HP were used separate N-6His-CBF-sc Fv.The purity of the purified N-6His-CBF-sc Fv was 90%.Identified by ic ELISA,anti-carbofuran sc Fv maintains antigen binding activity,IC（50） value was 63.80 ng/m L,which could be used for the further study.

关 键 词：克百威 单链抗体 分泌表达 纯化 鉴定 

分 类 号：TS201.2[轻工技术与工程—食品科学]