摘要
目的:通过将筛选的UL29shRNA表达质粒与抗病毒药物阿昔洛韦(ACV)的抗病毒效果及细胞毒性进行比较,探讨RNA干扰在细胞水平上对HSV-2病毒的抑制效果。方法:将筛选的干扰效果最好的表达质粒UL29-shRNA1641作为RNA干扰组与抗病毒药ACV进行对HSV-2的抑制效果的比较研究,分为RNAi组、ACV组和RNAi+ACV组。采用实时荧光定量PCR检测UL29mRNA的相对表达量,Karber法检测细胞上清液中病毒滴度的变化、蛋白印迹法(Western blot)检测ICP8的相对表达量,对RNAi与ACV抑制效果进行比较,通过WST-1(Water-Soluble Tetrazolium-1)法对质粒转染复合物(质粒+转染试剂)和ACV的细胞毒性进行比较。结果:RT-PCR检测三组mRNA的相对表达水平,其中RNAi组UL29基因相对表达量高于ACV组(P<0.05);RNAi+ACV组UL29基因相对表达量明显低于RNAi组和ACV组(P<0.05)。Karber法检测三组细胞上清液病毒滴度表明,RNAi组病毒滴度高于ACV组(P<0.05),RNAi+ACV组的病变病毒滴度明显低于RNAi组和ACV组(P<0.05)。Western blot检测ICP8相对表达量,ACV组的ICP8(UL29编码的单链DNA结合蛋白,主要作用是在HSV-2 DNA复制过程中与复制叉产生了单链DNA结合,防止其重新配对形成ds DNA或被核酸酶降解)相对表达量比RNAi组低(P<0.05)。RNAi+ACV组ICP8相对表达量降低最为明显,与ACV组和RNAi组相比有显著差异(P<0.05)。WST-1法对转染复合物与ACV的细胞毒性进行比较,其中RNAi中质粒和转染试剂对细胞的毒性明显小于ACV。结论:在RNAi与ACV对HSV-2抑制效果的比较中,ACV要好于RNAi,两者联合使用的抑制效果好于单独使用RNAi或ACV。在抑制效果都较好的情况下比较,RNAi中使用的质粒与转染试剂对细胞的影响比ACV小。
Objective： Aim to construct short hairpin RNA（shRNA） recombinant expression vector for herpes simplex virus typeⅡ（HSV-2） UL29 gene and observe the comparison of inhibition effect to HSV-2 of UL29 shRNA expression vector and ACV,in order to study the significance of RNA interference in the field of antiviral.Methods： The best sequence was selected from four UL29 shRNA sequences and transfected into HEK293 cells by liposome.The inhibition rate of this interference group was compared with acyclovir（ACV）.Virus was amplified and collected.The transcription level of UL29 in each group was estimated using real-time fluorescent quantitative PCR（RT-PCR）.The virus titer in each group was measured by Karber method.The expression level of ICP8 protein（UL29 coding single-stranded DNA binding protein）in each group was detected with Western blotting.The cytotoxicity of transfection complexes and ACV were tested with WST-1 method.Results： The result of Karber method shows that the viral titer（TCID50） is 10-5.375 /0.1 m L.The transcription level of UL29 in each group was estimated using RT-PCR,the relative expression of RNAi group,ACV group and RNAi ＋ ACV group are reduced in varying degrees（P〈0.05）.The result of Kaber assay showed that virus titers of RNAi group,ACV group and RNAi ＋ ACV group are reduced in varying degrees.Using Western blotting to detect the ICP8 has revealed that UL29 shRNA group,ACV group and RNAi ＋ ACV group can reduced protein expression in varying degrees.Among them,the most obvious effect is RNAi ＋ ACV group,protein expression is significantly reduced.There are significant differences compared with RNAi group and ACV group（P〈0.05）.The cytotoxicity of transfection complexes and ACV were tested with WST-1,the result showed that the cell lesions rates of transfection complexes is much less than ACV.Conclusion： With ACV for HSV-2 listed in RNAi inhibiting effect comparison,ACV is better than RNAi.As well as,using the combination therapy of RNAi and ACV has
出处
《现代生物医学进展》
CAS
2016年第11期2057-2060,2086,共5页
Progress in Modern Biomedicine
基金
贵州省科学技术基金项目(黔科合LH字[2014]7581号)
关键词
RNA干扰
Ⅱ型单纯疱疹病毒
UL29基因shRNA
阿昔洛韦
RNA interference
Herpes simplex virus type Ⅱ（HSV-2）
UL29 gene small hairpin RNA（UL29shRNA）
Aciclovir（ACV）
