盐酸克伦特罗生物素化单链抗体在大肠埃希氏菌中的表达  

Expression of Clenbuterol Biotinylated ScFv in Escherichia Coli

作　　者：马巍娜[1] 刘雪林[2] 宋宏彬[2] 沈建良[1] 黄友章[1] 刘毅[1] 向丹[1] 

机构地区：[1]中国人民解放军海军总医院血液科,北京100037 [2]中国人民解放军军事医学科学院十所,北京100039

出　　处：《现代检验医学杂志》2016年第4期44-46,50,共4页Journal of Modern Laboratory Medicine

基　　金：国家863计划科学基金资助项目(项目编号:2006BAK02A09)

摘　　要：目的 提取盐酸克伦特罗（CLB）生物素化抗体片段蛋白进行表达,为下一步纯化及农兽药品快速检测奠定基础。方法 确定一株生物素化CLB噬菌体抗体 “pHEN1-BHNS-CLB1”对其进行蛋白表达,即从噬菌粒“PHEN1-BHNS-CLB1”切下scFv基因片段,亚克隆至能引导可溶性表达的载体pCANTAB5E上,完成了重组质粒的构建,将所构质粒转化大肠埃希氏菌感受态细胞BL21。命名“5E-BHNS-CLB1”,并对“5E-BHNS-CLB1”进行PCR及酶切鉴定后,测序分析。结果 ELISA结果证明其具有较好的亲和性,竞争抑制ELISA、与gp120蛋白及三聚氰胺的交叉反应ELISA结果均证明其具有很好的特异性,片段大小与预想目的片段大小一致,SDS-PAGE及Western blotting的结果显示,其分子量大小与目的一致,并能与抗E-tag标签抗体结合显色。结论 说明表达的蛋白能与表达载体5E后带有E-Tag标签的短肽作用,含有E-Tag标签,是我们要表达的目的蛋白,为下一步纯化蛋白创造了条件。Objective to extract clenbuterol hydrochloride （CLB） biotiny/ated antibody fragments of protein expression,puri-fication andlay the foundation for the rapid detection of pesticide and veterinary drug next. Methods Determined biotinylated phage antibodies to CLB ＂pHEN1 BHNS CLBI＂ for protein expression. The scFv gene was cut from the phagemid ＂PHEN1-BHNS-CLBI＂, cloned into the vector pCANTAB5E can guide the soluble expression, completed the construction of recombinant plasmid,the plasmid was transformed into E. coli competent cells BL21. Named ＂5E-BHNS-CLBI＂, and ＂SE- BHNS-CLBI＂ by PCR and enzyme digestion,sequencing. Results The ELISA results proved that it had better affinity,com- petitive inhibition of ELISA, ELISA and gpl20 cross reaction results indicated that the protein and melamine had good speci- ficity,the scFv gene was cut from the phagemid ＂PHENI-BHNS-CLBI＇, cloned into the vector pCANTABSE could guide the soluble expression,completed the construction of recombinant plasmid,the plasmid was transformed into E. coli competent cells BL21. Named ＂SE-BHNS-CLBI＂,and ＂5E-BHNS-CLBI＂ by PCR and enzyme digestion,sequencing, fragment size and expected fragment size consistent. SDS-PAGE and Western blotting results showed that consistency in their molecular size and purpose,and with the label of anti e-tag antibody combined with color. Conclusion Protein expression and expression vector 5E with e-tag label short peptide,containing the E-tag label is we want to table of the target protein,as a purified protein to create the conditions.

关 键 词：噬菌体表面展示技术 筛选 盐酸克伦特罗 生物素 表达 

分 类 号：R378.21[医药卫生—病原生物学] R392.11[医药卫生—基础医学]