建立人颗粒溶素基因表达含量荧光定量PCR检测方法的研究

Establishment of Fluorogenic Quantitative Method for the Measurement of GNLY Gene Expression

目的建立人颗粒溶素（granulysin.GNLY）mRNA荧光定量的检测方法，构建克隆载体pMD18-T-GNLY作为定量模板，在ABI 7900HT型实时荧光定量PCR检测仪上通过荧光强度达到一定阈值时的循环数来定量起始模板，来建立实时荧光定量RT-PCR方法，并检测了40名正常健康献血者外周血中GNLY的表达。结果本方法的动态检测范围为10^3～10^9拷贝／μg RNA（r^2≥0.996），内参18s rRNA的动态检测范围为10^3～10^8拷贝/μg RNA（r^2≥0.996）；低值的批内、批间的重复性分别为10.33％和17.32％，高值的批内、批间的重复性分别为6．41％和8．46％；而40名正常健康人外周血的PBMCs中均有GNLY mRNA的表达，范围为3.12×10^6～4．32×10^7拷贝／μgRNA，均值为（2．0±1．3）×10^7拷贝／μg RNA。结论成功建立了人GNLY基因表达含量的荧光定量检测方法，检测结果可用绝对拷贝数表示，定量准确可靠。GNLY mRNA荧光定量测定方法的建立为研究GNLY mRNA表达水平与感染性疾病免疫机制的关系奠定了基础。

Objective To establish a specific fluorogenic quantitative method for detecting the expression of GNLY gene in peripheral blood mononuclear cells （PBMCs） in 40 normal people. Methods A real-time quantitative reverse transcription（RT）-PCR was set up, based on fluorescent TaqMan methodology. In this method, a prokaryotic expressing vector pMD18-T-GNLY was constructed as a standard plasmid and RNA quantification is based on the threshold cycle （Ct） values when using ABI 7900HT Sequence Detection Systems to examine the specific expression of GNLY in 40 normal people. Results The dynamic range of the assay was 10^3～10^9 copies/μg RNA, and that of endogenous standards was 10^3～10^8 copies/μg RNA. The relationship between Ct values and log starting concentration was linear（r^2≥0.99）. The intra-assay precision and inter-assay precision for a low value is 10.33% and 17.32% respectively, and the intra-assay precision and inter-assay precision for a high value is 6.41% and 8.46% respectively. In 40 normal people, the GNLY mRNA expression ranges from 3.12×10^6～4.32×10^7 copies/μg RNA and the mean value is 2.0±1.3×10^7 copies/μg RNA. Conclusion GNLY gene expression detected with FQ-PCR had an accurate speedy results. GNLY mRNA may be used as a valuable marker for investigating the relationship between GNLY mRNA expression and infectious diseases.