中文摘要：

目的：克隆人PLCE1基因,构建PLCE1 rs2274223和rs3765524 GT（PLCE1 Minor）和AC（PLCE1 Major）单体型真核表达重组载体,研究PLCE1基因多态性及单体型与基因表达的相关性。方法：利用重叠延伸PCR、In-Fusion技术构建PLCE1真核表达载体;基于同源重组的双点突变技术改造目的基因;利用基因转染、实时定量PCR和蛋白印迹等技术实现PLCE1表达载体在真核细胞中的过表达及表达水平的鉴定。结果：成功扩增并获得了全长6 927bp的人PLCE1 cDNA并经过DNA测序证实,与NCBI数据库PLCE1参考序列（NM_016341）比较,发现编码区3 554～3 572bp处存在18bp连续碱基插入,其余序列基本匹配。成功构建了PLCE1 Minor和PLCE1 Major两种单体型重组真核表达载体,转染细胞揭示PLCE1 Minor型的mRNA转录和蛋白质表达水平均高于Major型。结论：发现了一种新的PLCE1 mRNA转录本,成功构建了2种单体型真核表达载体;发现PLCE1 rs2274223G-rs3765524T单体型能够促进自身mRNA和蛋白质表达,为进一步揭示PLCE1 SNPs与癌症易感性的关系奠定了基础。

英文摘要：

Objective： The aimed is to address the question of how PLCE1 gene polymorphism or genotypes affect its expression by cloning human PLCE1 gene and constructing eukaryotic expression vector of PLCE1rs2274223G-rs3765524T（ PLCE1 Minor） and PLCE1 rs2274223A-rs3765524C（ PLCE1 Major）. Methods：Overlap extension PCR（ SOE-PCR） and In-Fusion Technology were adopted to construct PLCE1 eukaryotic expression vector. The targeted gene was acquired by double point mutations based on homologous recombination. The expression of overexpressed PLCE1 by gene transfection was observed by real-time quantitative PCR and Western blotting. Results： The full-length human PLCE1 that contains 6 927 base pairs was acquired and confirmed by DNA sequencing. By comparing with the reference sequence（ NM_016341） in NCBI database,it was found to exactly matched the reference sequence except a 18 bp insertion in the region of 3 554 ~3 572 bp. Additionally,the recombinant plasmids of two PLCE1 haplotypes,PLCE1 Minor and PLCE1 Major,were constructed respectively. By transfection,it was found that the expression levels of mRNA and protein of PLCE1 Minor were significantly higher than that of PLCE1 Major. Conclusion： A new PLCE1 transcript variant was discovered and the eukaryotic expression vectors of two haplotypes of human PLCE1 were successfully constructed. It was found that PLCE1 rs2274223G-rs3765524 T promotes its mRNA and protein expression.These findings laid the foundation of revealing the correlation between PLCE1 single nucleotide polymorphisms（ SNP） and cancer susceptibility.