人干扰素诱导跨膜蛋白基因实时荧光定量PCR检测方法的建立

目的建立人干扰素诱导跨膜蛋白(Interferon induced transmembrane protein,IFITM)1、2、3基因实时荧光定量PCR检测方法。方法根据GenBank中登录的人IFITM1、2、3及β-actin基因序列,分别设计4对特异性引物,以质粒pVAX1-IFITM1、2、3和pMD18-T-β-actin为模板,分别进行PCR及实时荧光定量PCR扩增,优化实时荧光定量PCR反应体系及反应条件,绘制标准曲线及扩增曲线,建立实时荧光定量PCR检测方法,并验证该方法的重复性、灵敏度及特异性。结果各质粒的PCR及实时荧光定量PCR产物经琼脂糖凝胶电泳分析,均可见大小与理论值相符的特异性条带。IFITM1、2、3和β-actin的最佳荧光定量PCR引物浓度分别为0.2、0.5、0.4和0.5μl,最佳退火温度为55℃。各质粒标准曲线的循环数与起始模板拷贝数的相关性均较好(R2分别为0.998、0.990、0.990和0.995),扩增效率分别为106.284%、101.030%、104.929%和101.962%。IFITM1、2、3基因不同拷贝数的试验内CV为0.14%-0.72%,试验间CV为0.77%-1.58%;灵敏度分别为2.52×100、1.3×100和3.7×101copies;该方法未扩增出鸡肝脏及禽流感病毒H3N2 cDNA,可特异性扩增B型流感病毒cDNA,各标准质粒的溶解曲线约在同一温度出现1个单峰。结论已成功建立了IFITM1、2、3基因实时荧光定量PCR检测方法,该方法具有较高的重复性、灵敏度及特异性,为进一步研究IFITM1、2、3的功能及其基因表达与肿瘤发生发展的相关性奠定了基础。

Development of real-time fluorescent quantitative PCR assay for human interferon induced transmembrane protein gene

Objective To develop a real-time fluorescent quantitative PCR assay for human interferon induced transmembrane protein(IFITM)1,2 and 3 genes.Methods Four pairs of specific primers were designed according to the sequences of IFITM1,2,3 and β-actin(as a internal reference)genes in GenBank.Target genes were amplified by PCR and realtime fluorescent quantitative PCR using plasmids pVAX1-IFITM1,pVAX1-IFITM2,pVAX1-IFITM3 and pMD18-T-β-actin as templates separately,based on which standard and amplification curves were plotted by optimization of system and condition for real-time fluorescent quantitative PCR.The developed method was verified for reproducibility,sensitivity and specificity. Results The PCR and real-time fluorescent quantitative PCR products of various plasmids showed specific bands on agarose gel electrophoretic profile,of which the lengths were consistent with the theoretical values.The optimal primer concentrations for amplification of IFITM1,2,3 and β-actin genes by real-time fluorescent quantitative PCR were 0.2,0.5,0.4 and 0.5 μl respectively,while the optimal temperature for annealing was 55 ℃.The correlation coefficients of cycle numbers to original copy number of plasmids pVAX1-IFITM1,pVAX1-IFITM2,pVAX1-IFITM3 and pMD18-T-β-actin on standard curves were 0.998,0.990,0.990 and 0.995,while the amplification efficiencies were 106.284%,101.03%,104.929% and 101.962%,respectively.The CV values of intra-and inter-assays on IFITM1,2 and 3 genes were 0.14% ~ 0.72% and 0.77% ~ 1.58% respectively.The sensitivities of developed method for IFITM1,2 and 3 genes were 2.52×10 0, 1.3×10 0 and 3.7×10 1 copies respectively.Neither chicken liver nor avian influenza virus H3N2 cDNA was amplified by the developed method,while influenza virus type B was amplified.The melting curves of various standard plasmids showed a single peak at the same temperature.Conclusion The real-time fluorescent quantitative PCR assay for IFITM1, 2 and 3 genes,with high reproducibility,sensitivity and specificity,was developed successfully,which laid a foundation of further study on functions of IFITM1,2 and 3 genes as well as relationship between their expressions and onset and progress of various diseases.