摘要
采用PCR技术从pGEM-pCD8α重组质粒中克隆了pCD8α胞外区去信号肽基因片段,并构建原核表达载体pET-30a-pCD8α-ED,在不同IPTG浓度和时间下进行诱导表达.结果表明:SDS-PAGE表达出约25ku的融合蛋白,目的蛋白主要以包涵体的形式存在,IPTG最佳诱导浓度为0.5mmoL·L-1,诱导6h时表达量达到峰值;Western-blot结果显示,在25ku左右出现杂交条带,表明表达的融合蛋白能被抗HIS标签的单克隆抗体识别,说明目的蛋白在大肠杆菌中得到了成功表达.
In order to preparing more porcine CD8α-ED recombinant protein with biological activity,porcine CD8α molecule extracellular domain fragment（pCD8α-ED）was amplified by PCR method from the pGEM-T-pCD8α recombinant plasmid,the pCD8α-ED was cloned into pET-30a vector and expressed in Escherichia coli.Inducible expression was carried out in different concentrations of the IPTG and different inducible expression time.The results showed that the fusion protein was exists mainly in the form of inclusion bodies and with the molecular weight of 25 ku by SDS-PAGE analysis.The optimum inducible concentration of IPTG was 0.5 mmoL·L-1,and the optimum inducible time was 6 h.The analysis of Western-blot indicated that the hybridization band was appeared around in the 25 ku,and it showed that the fusion protein was identified by the monoclonal antibody of HIS label protein,so it was showed that the target protein was expressed successfully in the Escherichia coli.
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2010年第6期18-22,共5页
Journal of Gansu Agricultural University
基金
国家自然基金项目(30871884)
甘肃省农业生物技术专项(GNSW-2008-2)
甘肃省科技支撑计划(0804NKCA076)
关键词
猪CD8α分子
胞外区基因片段
原核表达
porcine CD8α gene
fragment of extracellular domain（pCD8α-ED）
prokaryotic expression
