Cloning and identification of a phospholipase gene from Vibrio mimicus

Jung-Hwa Kang , Jong-Hee Lee , Je-Hyeon Park , Sung-Hoi Huh , In-Soo Kong
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引用次数: 26

Abstract

The phospholipase gene phl was identified from Vibrio mimicus (ATCC33653) and sequenced. The entire open reading frame (ORF) was composed of 1410 nucleotides and encoding 470 amino acids. The phl was placed upstream of hemolysin gene (vmhA) with opposite direction of transcription. From the BLAST search program, the deduced amino acids sequence showed 74.4% identity with phospholipase gene (lec) from V. cholerae El Tor. The entire ORF of phospholipase gene was amplified by PCR and inserted into an Escherichia coli expression vector, pET22b(+) and introduced E. coli BL21(DE3). SDS–PAGE demonstrated that a protein corresponding to the phospholipase was overexpressed and migrated at a molecular mass of 53 kDa.

拟态弧菌磷脂酶基因的克隆与鉴定
从模拟弧菌(Vibrio mimicus, ATCC33653)中鉴定出磷脂酶基因phl,并对其进行了测序。整个开放阅读框(ORF)由1410个核苷酸组成,编码470个氨基酸。phl位于溶血素基因(vmhA)的上游,转录方向相反。从BLAST程序中,推导出的氨基酸序列与霍乱弧菌El - Tor的磷脂酶基因(lec)的同源性为74.4%。通过PCR扩增出磷脂酶基因的完整ORF,将其插入大肠杆菌表达载体pET22b(+)中,导入大肠杆菌BL21(DE3)。SDS-PAGE显示,磷脂酶对应的蛋白过表达并迁移,分子量为53 kDa。
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