摘要
以单增李斯特菌基因组DNA为模板,利用自行设计的引物,通过PCR法扩增出单增李斯特菌的iap基因。在iap基因的5'端和3'端分别引入EcoRⅠ和XhoⅠ2个酶切位点将其克隆到pMD-18T载体上,构建克隆载体pMD-18T-iap。经测序正确后,将iap基因克隆至表达载体pET-28a上构建表达质粒pET-28a-iap。在IPTG诱导下,携带pET-28a-iap的E.coliBL21(DE3)高效表达分子质量约为60kD的可溶性蛋白及包涵体形式的蛋白,其中可溶性蛋白占总p60蛋白含量的76.3%左右。诱导表达的可溶性蛋白通过Ni2+亲和层析柱纯化得到纯度在95.6%以上的重组p60蛋白,其提取率为68.3%左右。
In this study,iap gene of Listeria monocytogenes was amplified by PCR using self-designed primers and genomic DNA of Listeria monocytogengs as the template.Through introducing EcoR Ⅰ and Xho Ⅰ sites to the 5 ＇and 3 ＇ ends of iap gene,iap gene was inserted into pMD18-T vector and recombinant pMD-18T-iap plasmid was constructed.After the verification of DNA sequence,the iap gene was subcloned into expression vector pET-28a to obtain pET-28a-iap expression plasmid.Through ITPG induction of pET-28a-iap,the recombinant p60 protein with relatively molecular mass of 60 kD was over-expressed in E.coli BL21（DE3）.The p60 protein was composed of soluble protein and inclusion protein and the amount of soluble protein reached up to 76.3%.The soluble protein was purified by Ni2＋-chelating affinity column chromatography with an extraction rate of 68.3%,and the purity of recombinant protein was 95.6%.These results will provide a reference for further studies on structure and functions of p60 protein.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第11期157-161,共5页
Food Science
基金
国家大学生创新性实验计划资助项目(200837)
