人亲磷脂酸磷脂酶A1基因的克隆与序列分析及真核表达载体的构建

Cloning and sequence analysis of human phosphatidic acid-preferring phospholipase A1 gene and construction of the eukaryocyte expression vector of the enzyme

目的克隆人亲磷脂酸磷脂酶A1(PA-PLA1)基因,分析该酶基因及其蛋白质的序列特征并构建PA-PLA1基因真核表达载体,为该酶生理功能的研究提供实验依据。方法从人的肾脏组织中提取总RNA,逆转录制备cDNA。扩增人PA-PLA1基因并克隆测序。以Vector NTI 8.0、DNASTAR、ORF finder分析所克隆的人PA-PLA1基因及其蛋白质序列。构建真核表达载体pcDNA3.1-PA-PLA1,测序证实后转染小鼠分泌胰岛素细胞株MIN6,以Western blotting检测PA-PLA1蛋白的表达水平。结果人PA-PLA1基因mRNA全长为2 923 bp,编码区长2 238 bp,编码1个由745个氨基酸残基组成的蛋白多肽。序列比对表明所克隆的人PA-PLA1基因与牛PA-PLA1基因同源,并与KIAA0725p和p125基因高度相似,三者在脂肪酶家族中形成亚家族,但KIAA0725p蛋白和p125蛋白具有常见的脂肪酶活性中心保守序列GX-S-XG,而PA-PLA1的该序列为SX-S-XG。真核表达载体pcDNA3.1-PA-PLA1构建正确并在所转染的MIN6细胞中过表达PA-PLA1蛋白。结论 PA-PLA1、KIAA0725p蛋白和p125蛋白在脂肪酶家族中形成亚家族,但PA-PLA1具有独特的脂肪酶活性中心保守序列SX-S-XG。所构建的PA-PLA1基因真核表达载体能在MIN6细胞中过表达小鼠PA-PLA1蛋白。该研究结果将为PA-PLA1生理功能的研究提供实验依据。

【Objectives】 To clone human phosphatidic acid-preferring phospholipase A1（PA-PLA1） gene, analyze the characters of the sequences of the PA-PLA1 gene and its protein,and construct a eukaryocyte expression vector of PA-PLA1 gene,so that to provide data for further study of the function of PA-PLA1. 【Methods】 Total RNA was extracted from human kidney tissue and the cDNA was prepared by reversed transcription.Human PA-PLA1 gene was amplified,cloned and sequenced.The sequences of the human PAPLA1 gene cloned and its protein were analyzed using Vector NTI 8.0,DNASTAR and ORF finder.The eukaryocyte expression vector of mouse PA-PLA1 gene pcDNA3.1-PA-PLA1 was constructed and the vector was confirmed by DNA sequencing.The expression of PA-PLA1 protein was detected with Western blotting.【Results】 The full-length cDNA sequence of human PA-PLA1 gene was 2923 bases in length and contained an open reading frame of 2238 nucleotides encoding a 745-amino acid protein.The gene sequence of humanPA-PLA1 was highly homologous to that of bovine and resembled that of KIAA0725p and p125.PA-PLA1, KIAA0725p and p125 formed a subfamily in lipase family,but KIAA0725p and p125 proteins contained a conserved sequence GX-S-XG,which is present commonly in the active center of lipase and in contrast,PAPLA1 contained a conserved sequence SX-S-XG.The eukaryocyte expression vector pcDNA3.1-PA-PLA1 was accurately constructed and was able to over-express mouse PA-PLA1 protein in MIN6 cells transfected with the vector pcDNA3.1-PA-PLA1.【Conclusion】 PA-PLA1,KIAA0725p and p125 can form a subfamily in lipase family,but PA-PLA1 has the unique lipase conserved sequence in active center GX-S-XG.The constructed eukaryocyte expression vector pcDNA3.1-PA-PLA1 is able to over-express mouse PA-PLA1 protein in mouse insulin-secreting cell line MIN6.This study will provide data for further study of the physiological function of PA-PLA1.