荧光定量PCR用于hTRIMCyPA抑制HIV-1的研究

Fluorescence quantitative PCR assay for inhibition of HIV-1 by hTRIMCyPA

目的:建立相对荧光定量PCR测定HIV-1晚期反转录产物方法,并以其评价hTRIMCyPA对HIV-1的抑制作用。方法:选择HIV-1的LTR区和HIV-1Gag之间的序列设计晚期反转录产物引物,以GAPDH基因为内参基因,建立SYBR GreenⅠ相对荧光定量PCR测定HIV-1晚期反转录产物的体系,并用来评价hTRIMCyPA重组蛋白对HIV-1晚期反转录产物的影响。结果:在建立的荧光定量PCR体系中,晚期反转录产物和内参GAPDH标准曲线的斜率分别为-3.381、-3.289。相关系数R 2分别为0.9929、0.9937,均>0.99,表明二者的线性较好,晚期反转录产物基因及参照基因GAPDH基因扩增效率相同,可用于相对定量分析。hTRIMCyPA表达组HIV-1相对晚期反转录产物量明显少于空载体组。结论:本研究建立的定量HIV-1特异性反转录产物相对荧光定量PCR检测方法简单、高效和成本低廉,并且hTRIMCyPA对HIV-1反转录具有明显抑制作用。

Objective:To establish a relative fluorescence quantitative PCR method for the determination of late reverse transcription products(late RT)and use this method to study the inhibitory effect of hTRIMCyPA on HIV-1.Methods:The sequence between the LTR region of HIV-1 and the Gag conserved region was selected to design the primers for the late RT to establish the SYBR GreenⅠrelative fluorescence quantitative PCR system with the GAPDH as a reference for the determination of the late RT of HIV-1,and to evaluate the effect of hTRIMCyPA protein on the late RT of HIV-1.Results:The correlation coefficients R 2 of the late RT and the GAPDH standard curve in the established fluorescence quantitative PCR system were 0.9929 and 0.9937 with the slope were-3.381,-3.289,respectively.The correlation coefficient R 2>0.99 of the two standard curves indicates that the two are well linear,and the amplification efficiency of the late RT and the GAPDH gene is the same,which can be used for relative quantitative analysis.The amount of HIV-1 late RT in hTRIMCyPA gene expression group was significantly lower than that in empty vector group.Conclusion:The relative fluorescent quantitative PCR detection method established in this study has the advantages of simplicity,efficiency,and low cost,hTRIMCyPA significantly inhibits HIV-1 reverse transcription.