蜡样芽胞杆菌深圳株754-1 PLC基因的克隆及表达

CLONING AND EXPRESSION OF THE PHOSPHOLIPASE C GENE OF BACILLUS CEREUS

以本实验室分离筛选的高产PLC的蜡样芽胞杆菌深圳株754-1为供体菌,以大肠杆菌DH5α和BL21(DE3)为受体菌,构建高效表达胞内PLC的工程菌。以754-1菌基因组为模板,经PCR扩增,将得到的PLC基因连接到T载体上,转入大肠杆菌DH5α。经Amp抗性筛选的阳性克隆子提取重组质粒,双酶切,回收含PLC基因的片段并将其克隆到pGEX-KG载体,获得重组质粒pGEX-KG-PLM,该重组质粒转化大肠杆菌BL21(DE3)。用IPTG诱导表达,SDS-PAGE分析,在分子量为38kd处有一条明显的表达带。

Phospholipase C gene from the donor named Bacillus cereus 754-1 of Shenzhen, was transformed into the host of E. coli and the engineering strain expressing Phospholipase C was constructed. The DNA sequence coding for Phospholipase C, with the genomic DNA of Bacillus cereus 754-1 of Shenzhen as template, was cloned and ligated into pMD18-T then transformed into E. coli DH5α. Recombinated pasmids pMD18-PLM contained in positive clone with Amp resistance were extracted and digested by double digestion, the digested DNA containing phospholipase C gene was purified and cloned into the vector pGEX-KG . In this way, the recombinated pasmids pGEX-KG-PLM was constructed and expressed in E. coli BL21 （ DE3 ）. The result showed pGEX-KG-PLM can secrete a protein with the size of 38kd by SDS -PAGE analysis with IPTG as induced agent.