关键词: 分子克隆, 基因表达, PCR, 大肠杆菌

Abstract: In this study, mature peptide coding region YT_m-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-YT_m-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 YT_m-MAP30 in the recombinant plasmid pET-YT_m-MAP30 was correct. SDS-PAGE analysis show ed 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.

Key words: molecular cloning, gene expression, PCR, Escherichia coli