摘要
在分析水貂肠炎病毒VP2基因主要抗原位点分布的基础上,设计了1对特异性引物,克隆一段长为846 bp,编码主要抗原位点的基因片段。将该基因片段定向插入表达载体PET-32a中,转化BL21(DE3)菌株,IPTG诱导实现高效表达。诱导表达比较实验结果表明,在37℃,IPTG浓度为1.0 mM,诱导时间为6 h,诱导表达达到最大效率。重组蛋白以包涵体形式存在,大小约为50 KD,应用兔抗水貂MEV抗体,经Western-blotting验证该重组蛋白具有MEV的抗原性,可为今后抗MEV单克隆抗体制备及相关免疫学检测方法的建立提供良好的抗原物质。
We amplified a 846 bp nucleotide fragment encoding the main antigenic site of mink enteritis virus VP2 protein by polymerase chain reaction（PCR） assay after analyzing the epitope domain of the VP2 gene.The fragment was cloned into PET-32a vector and transformed into Escherichia,coli BL21（DE3）.Recombination protein was efficiently expressed after being induced with IPTG.The results of the comparative experiments for expression inducement showed that the protein expression was of the highest efficiency in the condition of 1.0 mM IPTG,at 37℃for 6 hours.SDS - PAGE analysis showed that the protein was about 50 KD and was expressed in the form of an inclusion body.The result of western - blotting revealed that the recombination protein reacted well with anti - MEV serum.All results indicated that the recombination protein can be valuable in providing a useful antigen for preparation of anti - MEV monoclonal antibody and related immunological detection method development.
关键词
水貂肠炎病毒
VP2基因
原核表达与鉴定
Mink enteritis virus
VP2 gene
Prokaryotic expression and identification
