摘要
根据GenBank中牛分枝杆菌的基因序列,设计合成一对扩增CFP-10基因完整ORF的特异性引物,以pET32a(+)为载体构建重组表达载体CFP-10/pET32a(+)。重组表达载体转化大肠杆菌BL21(DE3),经0.9mmol/L IPTG37℃诱导3h后表达,收集菌体并裂解,裂解后上清和沉淀经SDS-PAGE分析,结果表明融合蛋白分子量分别为30kD,表达产物以可溶性存在于上清中,后经Ni-NTA吸附柱方法对目的蛋白进行纯化,经薄层凝胶扫描分析,纯化后的His融合蛋白纯度达95%,该CFP-10表达蛋白可作为体外诊断抗原,为进一步建立检测牛分枝杆菌的方法奠定基础。
According to Mycobacterium bovis gene sequences in GenBank, a pair of primers was designed and synthesized to amplify the whole ORF （open reading frame） of the CFP-IO （10 kD culture filtrate protein） genes from Mycobctcterium boris （M. boris） C68001 strain. The pET32a（＋） was used as a vector to construct recombinant expression vector CFP- 10/pET32a （＋）. The recombinant expression vector was transformed into E. coli BL21 （DE3）, and expressed under the induction of 0.9 mmol/L IPTG 37~C for 3 hours. The supernatant and sediment, which was from collected and supersonic thallus, were analyzed by SDS-PAGE and result showed that the molecular weight of fusion protein was 30 kD. The expression product is soluble on the supernatant and the purity of His fusion protein reached 95% after being purified by Ni-NTA adsorptive column method and gel thin layer scanning analysis. The CFP-10 expression protein can be used as an in vitro diagnostic antigen, and moreover as a cornerstone for the detection of Mycobctcterium bovis.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2012年第4期360-365,共6页
Genomics and Applied Biology
基金
广西特聘专家专项经费资助项目(2011B020)
新世纪百千万人才工程国家级人选专项基金(945200603)
公益性行业(农业)科研专项(201103008)
广西壮族自治区科技攻关项目(桂科转0626001-5-1)
广西壮族自治区水产畜牧局科研计划(桂鱼牧科[08]283-20和桂鱼牧科09254-19
18)共同资助
