苦瓜核糖体失活蛋白MAP30基因的克隆及原核表达

以载有银田牌长白苦瓜MAP30基因(YT-MAP30,Genebank accession No:DQ643968)的质粒DNA(pMD-YT-MAP30)为模板,PCR扩增其成熟肽编码区YTm-MAP30,与原核表达载体pET-30a(+)连接构建重组表达质粒pET-YTm-MAP30,酶切和测序鉴定后转化大肠杆菌BL21(DE3)PlysS感受态细胞,菌落PCR筛选鉴定阳性菌落。30℃培养阳性菌落,IPTG诱导表达不同时间,其表达产物用SDS-PAGE检测并进行可溶性分析。DNA测序结果表明重组质粒pET-YTm-MAP30中YTm-MAP30的序列和插入位点正确。SDS-PAGE分析结果显示IPTG诱导4 h后在35 kD处出现一条增加的表达蛋白条带,且以可溶形式表达。为应用原核表达系统生产MAP30蛋白提供了实验依据。

Cloning and Prokaryotic Expression of MAP30 Gene of Balsam Pear

In this study,mature peptide coding region YTm-MAP30 was amplified using plasmid DNA(pMD-YT-MAP30) which carries MAP30 gene of Yintian brand balsam pear as template,then the amplified sequence was cloned into prokaryotic expression vector pET-30a(+) and recombinant expression plasmid pET-YTm-MAP30 was constructed.The positive recombinant vector were transformed into E.coli BL21(DE3)PlysS competent cells after identification of restriction enzyme digestion and sequencing.Positive colonies were screened using colony PCR and cultured at 30 ℃,and then its expression was induced by IPTG for different time.The expressed products were analyzed by SDS-PAGE,and dissolving quality was analyzed by supersonic.The result of DNA sequencing showed that the sequence and cloning site of YTm-MAP30 in the recombinant plasmid pET-YTm-MAP30 was correct.SDS-PAGE analysis showed that a strengthened protein band was found in 35 kD protein marker after the recombinant bacteria were induced by IPTG for 4 hours,and the expressed protein was soluble.This study provided a foundation for the production of recombinant MAP30 protein using prokaryotic expression system.