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目的 通过构建花生四烯酸12-脂氧合酶(ALOX-12)基因(ALOX-12基因)启动子区域rs3840880位点不同基因型的双荧光素酶表达质粒,分析此单核苷酸多态性(single nucleotide polymorphisms,SNP)对ALOX-12基因启动子活性的影响及其对基因表达的影响,阐明功能性SNP调控结核病易感的分子机制.方法 从全血样本中提取人全基因组,PCR扩增ALOX-12基因启动子区域包含rs3840880位点在内的约1700 bp的基因片段,构建pGL3-Basic-rs3840880G质粒,对此质粒进行定点突变得到pGL3-Basic-rs3840880 D放散型(DEL)质粒,分别转染人宫颈癌细胞株Hela细胞后使用双荧光报告酶检测系统(dual luciferase assay system)检测相对荧光值.结果 成功构建了ALOX-12基因启动子区rs3840880位点等位基因G的表达质粒pGL3-basic-G,采用定点突变技术成功将pGL3-basic-G质粒改造为pGL3-Basic-DEL质粒,测序验证后,两质粒其他序列完全相同.分别将此两个质粒转染Hela细胞并检测相对荧光比值[荧光值(F)/荧光?]后,发现pGL3-Basic质粒F/R=0.129,等位基因G的F/R=0.194,低于等位基因DEL质粒的F/R(0.274),采用单因素方差分析,二者之间的差异有统计学意义(F=25.09,P=0.001).结论 不同等位基因构建的质粒转染细胞后,荧光报告基因的表达差异具有统计学意义,故ALOX-12基因启动子区域的功能性SNPs确实能够影响启动子的活性,从而调控结核病的易感性.“,”Objective To construct different genotype dual luciferase expression plasmid expressed the rs3840880 loci of promoter region located in arachidonic acid lipoxidase-12 (ALOX-12) gene, and to analyze the effects of this single nucleotide polymorphisms (SNP) on ALOX-12 gene promoter activity and gene expression, in order to elucidate the molecular mechanism of functional SNP regulated tuberculosis susceptibility.Methods The 1700 bp fragments contained rs2840880 loci of promoter region located in ALOX-12 gene were amplified with human genome DNA extracted from whole blood samples by PCR.The pGL3-Basic-rs3840880 DEL plasmids was obtained from pGL3-Basic plasmid constructed with the PCR products and the original plasmid by site-directed mutagenesis and transfected into human cervical carcinoma cell line(Hela).The relative fluorescence values were detected by the dual luciferase assay system.Results The pGL3-Basic-DEL plasmid from pGL3-Basic-G plasmid containing the rs3840880 loci of promoter region located in the ALOX-12 gene by site-directed mutagenesis was obtained and sequenced successfully.The relative fluorescence ratio (F/R) were detected after two plasmids were transfected into Hela cell line.The fluorescence ratio of allele G (F/R=0.194) was lower than that of allele DEL (F/R=0.274), pGL3-Basic (F/R=0.129) with significant difference statistically (F=25.09,P=0.001).Conclusion The expression differences of fluorescence reporter gene in the plasmids constructed with different alleles and transfected into Hela cell line are significant statistically.It shows that functional SNP of ALOX-12 gene affect really the activity of promoter and regulate tuberculosis susceptibility.