犬细小病毒VP2基因在大肠杆菌中的高效可溶性表达

目的:犬细小病毒VP2基因在大肠杆菌中高效可溶性表达,进而为其特异单克隆抗体的制备奠定基础。方法:以犬细小病毒VP2基因重组质粒为模板,通过蛋白质分析软件PROSPECT分析,根据VP2基因所编码的氨基酸的亲水性和抗原性,把VP2基因分成不同区段,设计相应引物并扩增出相应片段。按常规方法克隆入pGEX-4T-2载体谷光甘肽-S-转移酶(GST)基因的下游,获得的重组质粒转化大肠杆菌BL21,用IPTG诱导目的基因片段的表达,经超声处理后SDS-PAGE电泳。所得可溶性蛋白再经间接ELISA、western Blotting鉴定。结果:K2和K4片段获得了良好的可溶性表达,其表达的GST融合蛋白的分子质量分别为38.3 KD和41 KD,ELISA、Western Blotting结果表明所表达的融合蛋白具有与CPV阳性抗体特异结合的良好抗原性。结论:犬细小病毒VP2基因在大肠杆菌中的获得了高效可溶性表达。

Prokaryotic Soluble Expression of Canine Parvovirus VP2 Gene

Objective:To solubly express the canine parvovirus VP2 gene efficiently in Bacillus coli,and lay a foundation for the preparation ofmonoclonal antibodies against VP2 protein.Methods:Protein analysis software-PROSPECT was used to divide VP2 into different sections according to hydrophile and antigen character of VP2 amino acids.Correspondent down-lead was designed to extend the section.According to the conventional method,they were sub-cloned into the downstream of GST gene in expression plasmid pGEX-4T-2.The recombinant vector was transformed into E.coli BL21,and GST fusion protein was expressed at the induction of IPTG. SDS-PAGE,indirect ELISA and Western Blotting were used to identify the protein.Results:The fusion proteins were 38.3 KDa and 41 KDa.Both fusion proteins had good antigenicity,which was confirmed by SDS-PAGE,indirect ELISA and Western Blotting.Conclu- sion:Canine Parvovirns VP2 gene was solubly expressed in E.coli.