短发夹RNA表达载体对2型单纯疱疹病毒UL54干扰效应的研究

发布时间：2018-04-23 21:16

本文选题：RNA干扰 + 短发夹RNA　；参考：《遵义医学院》2011年硕士论文

【摘要】：目的：针对2型单纯疱疹病毒(Herpes simplex virus type 2, HSV-2) UL54基因构建短发夹RNA (small hairpin RNA, shRNA)表达载体,采用脂质体转染法介导表达载体转入人胚胎肾293株(Human Embryonic Kidney cells, HEK293)细胞,在体外细胞水平上观察shRNA表达载体对HSV-2 UL54基因表达的影响,以及对HSV-2在HEK293细胞中复制的影响。方法：针对HSV-2 UL54基因筛选出5条拟干扰位点,构建5个抑制UL54基因的短发夹RNA的真核表达载体,分别命名为shRNA1、shRNA2、shRNA3、shRNA4 shRNA5,以及不针对任何序列的阴性表达载体shRNA NC。通过脂质体介导表达载体转染HEK293细胞,48小时后再接种HSV-2。采用荧光定量RT-PCR技术检测UL54mRNA的转录水平,终点滴定法检测细胞上清液中的病毒滴度。结果：(1)酶切和测序鉴定重组表达载体shRNA1、shRNA2、shRNA3、shRNA4、shRNA5、shRNA NC构建成功；(2)荧光定量RT-PCR结果显示,与空白组(未转染重组表达载体),shRNA1、shRNA4、shRNA5均不同程度降低UL54 mRNA的表达水平(P0.05)。(3)终点滴定法结果显示shRNA1、shRNA4、shRNA5可不同程度降低上清液中的病毒滴度((P0.05)。结论：成功构建shRNA1、shRNA2、shRNA3、shRNA4、shRNA5重组表达载体,shRNA1、shRNA4、shRNA5能在体外细胞水平上不同程度的干扰HSV-2 UL54基因表达,抑制HSV-2在HEK293细胞中复制,为进一步利用RNAi技术抗HSV-2的研究提供参考数据。
[Abstract]:Objective: to construct the expression vector of short hairpin RNA small hairpin RNAs (shRNAs) targeting herpes simplex simplex virus type 2 (HSV-2) UL54 gene and transfer them into human Embryonic Kidney cells (HEK293) cells by liposome transfection. The effect of shRNA expression vector on the expression of HSV-2 UL54 gene and the replication of HSV-2 in HEK293 cells were observed in vitro. Methods: five pseudo interference sites were screened from HSV-2 UL54 gene, and 5 eukaryotic expression vectors of short hairpin RNA were constructed, named shRNA1hRNA2hRNA2hRNA3hRNA4 shRNA5, and negative expression vector shRNA nc. which did not target any sequence. HEK293 cells were transfected with liposome mediated expression vector for 48 hours and then inoculated with HSV 2. The transcriptional level of UL54mRNA was detected by fluorescence quantitative RT-PCR and the titer of virus in supernatant was detected by end-point titration. Results the recombinant expression vector shRNA1, shRNA2, shRNA3, shRNA4, shRNA5, shRNA5, was successfully constructed by restriction endonuclease digestion and sequencing. The results of titration showed that shRNA1shRNA4hRNA5 could decrease the titer of UL54 mRNA in the supernatant to a different extent, compared with the blank group (P 0.05, P 0.05, P 0.05, P 0.05). The results of titration showed that shRNA1shRNA4hRNA5 could decrease the virus titer (P0.05) in the supernatant. Conclusion: the recombinant expression vector of shRNA1hRNA2shRNA2hRNA3hRNA4hRNA4hRNA5 can interfere with the expression of HSV-2 UL54 gene in vitro and inhibit the replication of HSV-2 in HEK293 cells, which provides reference data for further research on anti- by RNAi technique.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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