NLRP1基因启动子区荧光素酶报告载体的构建与鉴定

Construction and identification of luciferase reporter gene vectors containing two haplotypes DNA of human NLRP1 gene promoter region

目的构建人NLRP1(nucleotide-binding,leucine-rich repeat pyrin domain containing protein 1)基因启动子区SNPrs878329位点两种单倍体荧光素酶报告基因载体。方法分别以SNP rs878329的GG和CC基因型的人基因组DNA为模板,用PCR法扩增出包含该位点的长476 bp的NLRP1基因启动子区目的片段,用KpnⅠ/BglⅡ双酶切,切胶回收后分别与同样双酶切的荧光素酶报告基因载体PGL3-promoter相连接,构建pGL3-promoter-G和pGL3-promoter-C两个表达质粒,并测序验证其DNA序列。结果成功构建了人类NLRP1基因启动子SNP rs878329的两种纯合子基因型(GG型和CC型)的荧光素酶报告载体,且测序得以证实。结论成功构建出的荧光素酶报告载体,为研究NLRP1基因启动子SNP rs878329能否调控NLRP1基因表达提供了基本实验条件。

This study is aimed to construct a luciferase reporter gene vector containing two different haplotypes DNA on SNP rs878329 of human NLRP1 gene promoter region.The 476 bp DNA fragments of NLRP1 promoter region including the SNP rs878329 were obtained by polymerase chain reaction（PCR） based on human genome DNA from the subjects with the GG/CC genotypes.Then the DNA fragments were digested by restriction endonucleases KpnⅠ and BglⅡ.After fragment recovery,those fragments were ligated to pGL3-promoter vectors,which had digested by restriction endonucleases KpnⅠ and BglⅡ as well,to construct recombinant plasmids pGL3-promoter-G and pGL3-promoter-C.By sequencing,we confirmed that the two recombinant plasmids（pGL3-promoter-G and pGL3-promoter-C） containing two haplotypes DNA of human NLRP1 promoter region were obtained.These expression vectors constructed in our study will be important tools for exploring the relation between the SNP rs878329 and the expression of NLRP1 gene.