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

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。

Expression of Clenbuterol Biotinylated ScFv in Escherichia Coli

Objective to extract clenbuterol hydrochloride(CLB)biotinylated antibody fragments of protein expression,purification andlay the foundation for the rapid detection of pesticide and veterinary drug next.Methods Determined biotinylated phage antibodies to CLB“pHEN1 BHNS CLB1” for protein expression.The scFv gene was cut from the phagemid “PHEN1-BHNS-CLB1”,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-CLB1”,and “5E-BHNS-CLB1” by PCR and enzyme digestion,sequencing.Results The ELISA results proved that it had better affinity,competitiveinhibition of ELISA,ELISA and gp120 cross reaction results indicated that the protein and melamine had good specificity,the scFv gene was cut from the phagemid “PHEN1-BHNS-CLB1”,cloned into the vector pCANTAB5E could guide the solubleexpression,completed the construction of recombinant plasmid,the plasmidwas transformed into E.coli competent cells BL21.Named “5E-BHNS-CLB1”,and“5E-BHNS-CLB1” by PCR and enzyme digestion,sequencing,fragment size and expected fragment size consistent.SDS-PAGE and Western blotting results showedthat consistency in their molecular size and purpose,and with the label of antie-tag antibody combined with color.Conclusion Proteinexpression and expression vector 5E with e-tag label short peptide,containingthe E-tag label is we want to table of the target protein,as a purified protein to create the conditions.