人乳头瘤病毒16型E2蛋白对巨噬细胞分泌活性及凋亡的影响

发布时间：2018-03-25 04:15

本文选题：HPV16　切入点：E2蛋白　出处：《南华大学》2011年硕士论文

【摘要】：目的：构建人乳头瘤病毒16型（HPV16）E2蛋白及其N端结构域（TAD）和C端结构域（DBD）与绿色荧光蛋白（GFP）融合蛋白真核表达载体，观察融合蛋白在Hela细胞或巨噬细胞（MΦ）中的定位。研究HPV16E2蛋白及其TAD、DBD对MΦ分泌TNF-α和IL-1β及细胞凋亡率的影响。方法： (1)用Primer5.0引物设计软件设计引物，聚合酶链反应（PCR）扩增HPV16E2及其TAD、DBD基因。PCR产物经双酶切后与pEGFP-C1载体相应酶切位点连接，转化E.coli JM109，经酶切和测序鉴定，筛选阳性克隆，构建真核表达载体pEGFP-C1/HPV16E2、pEGFP-C1/TAD及pEGFP-C1/DBD。 (2)将质粒pEGFP-C1、pEGFP-C1/E2、pEGFP-C1/TAD、pEGFP-C1/DBD各0.5μg分别转染Hela细胞或MΦ。36h后，倒置荧光显微镜下观察GFP-HPV16E2、GFP-HPV16E2TAD、GFP-HPV16E2DBD在Hela细胞或MΦ中的分布及定位。 (3)用佛波酯（PMA）诱导THP-1为MΦ；将MΦ分为空白组、GFP组、GFP-E2组、GFP-E2TAD组及GFP-E2DBD组。除空白组外，每组分别转染0.5μgpEGFP-C1、pEGFP-C1/E2、pEGFP-C1/TAD、pEGFP-C1/DBD。转染48h后，取细胞培养上清液, ELISA测定TNF-α和IL-1β的含量；收集细胞，流式细胞术（FCM）检测MΦ凋亡。结果： (1) PCR产物分别连接至pEGFP-C1载体上后，双酶切及测序结果表明目的片段与HPV16E2、TAD、DBD基因正确连接入pEGFP-C1。真核表达载体pEGFP-C1/E2、pEGFP-C1/TAD及pEGFP-C1/DBD分别转染Hela细胞或MΦ后，GFP-DBD融合蛋白定位于细胞核内，GFP-TAD定位于细胞浆，GFP-E2、GFP在细胞核、浆内均有表达，但GFP-E2表达组细胞核荧光亮度强于细胞浆。 (2)质粒转染MΦ48h后，细胞培养上清中TNF-α、IL-1β的含量GFP-E2组(321.83±22.00，，214.35±22.79)、GFP-TAD组(371.79±22.73，233.52±17.75)明显高于GFP组(265.74±28.74,167.60±18.68)和空白组(234.76±31.94,155.13±20.54)（P0.05），且GFP-TAD组与GFP-E2组TNF-α浓度差异有统计学意义（P0.05）；GFP-TAD组IL-1β含量略高于GFP-E2组，但差异无统计学意义（P0.05）；GFP-DBD(265.74±28.74，167.60±18.68)与GFP组或空白组TNF-α及IL-1β含量比较，差异无统计学意义（P0.05）。 (3)在MΦ凋亡率检测中，GFP-E2组(11.53±0.54)、GFP-TAD组(14.57±0.59)明显高于GFP组(4.53±0.37)或空白组(5.20±0.65)（P0.05），且GFP-TAD组MΦ凋亡率高于GFP-E2组（P0.05）；但GFP-DBD组(4.92±0.49)与GFP组或空白组比较，MΦ凋亡率差异无统计学意义（P0.05）。结论： (1)构建的真核表达载体pEGFP-C1/HPV16E2、pEGFP-C1/TAD、pEGFP-C1/DBD均能在Hela细胞或MΦ中表达，GFP-DBD定位于细胞核，GFP-TAD定位细胞浆。 (2) HPV16E2及其TAD即时高表达上调MΦ分泌TNF-α和IL-1β。 (3) HPV16E2及其TAD即时高表达可诱导MΦ凋亡。
[Abstract]:Aim: to construct the eukaryotic expression vector of the fusion protein of HPV16 E 2 and its N-terminal domain TAD and C-terminal domain of human papillomavirus 16 (HPV16) with green fluorescent protein (GFP). To observe the localization of the fusion protein in Hela cells or macrophages, and to study the effects of HPV16E2 protein and tadd DBD on the secretion of TNF- 伪 and IL-1 尾 and the apoptosis rate of M 桅. Methods:. 1) primers were designed with Primer5.0 primer design software. The PCR products were amplified by polymerase chain reaction (PCR). The PCR products were digested with the corresponding restriction sites of pEGFP-C1 vector and transformed into E.coli JM109. The positive clones were screened by restriction enzyme digestion and sequencing. The eukaryotic expression vector pEGFP-C1 / HPV16E2pEGFP-C1 / tat and pEGFP-C1 / DBDrespectively were constructed. (2) the plasmid pEGFP-C1pEGFP-C1 / E2pEGFP-C1 / pEGFP-C1 / TAD1 / pEGFP-C1 / DBD was transfected into Hela cells or M 桅. 36 h later, the distribution and localization of GFP-HPV16E2TADD in Hela cells or M 桅 were observed under inverted fluorescence microscope. (3) THP-1 was induced to be M 桅 by PMA, and M 桅 was divided into two groups: GFP-E2TAD group and GFP-E2DBD group. With the exception of blank group, each group was transfected with 0.5 渭 g pEGFP-C1 / E2pEGFP-C1 / E2pEGFP-C1pEGFP-C1 / DBD.After transfection, the supernatant of cell culture was taken, and the contents of TNF- 伪 and IL-1 尾 were measured by ELISA. Apoptosis of M 桅 was detected by flow cytometry (FCM). Results:. 1) the PCR product was ligated to the pEGFP-C1 carrier, The results of double enzyme digestion and sequencing showed that the target fragment was correctly ligated into pEGFP-C1.The eukaryotic expression vector pEGFP-C1 / E2pEGFP-C1pTAD and pEGFP-C1/DBD were transfected into Hela cells or M 桅, respectively. However, the nuclear fluorescence intensity of GFP-E2 expression group was stronger than that of cytoplasm. The content of TNF- 伪 IL-1 尾 in the supernatant of cell culture in GFP-E2 group was 321.83 卤22.00214.35 卤22.79TP-TAD group 371.79 卤22.73233.52 卤17.75, which was significantly higher than that in GFP group (265.74 卤28.74167.60 卤18.68) and blank group (234.76 卤31.94155.13 卤20.54P0.05), and there was a significant difference between GFP-TAD group and GFP-E2 group in TNF- 伪 concentration. The content of IL-1 尾 in GFP-TAD group was slightly higher than that in GFP-E2 group. However, there was no significant difference in the contents of TNF- 伪 and IL-1 尾 between the GFP group and the control group (265.74 卤28.74167.60 卤18.68). (3) the apoptosis rate of M 桅 in GFP-E2 group (11.53 卤0.54) was significantly higher than that in GFP group (4.53 卤0.37) or blank group (5.20 卤0.65), and the apoptosis rate of M 桅 in GFP-TAD group was higher than that in GFP-E2 group, but there was no significant difference between GFP-DBD group and GFP group or blank group. Conclusion:. The constructed eukaryotic expression vector pEGFP-C1 / HPV16E2pEGFP-C1 / TADPEGFP-C1 / DBD could express GFP-DBD in Hela cells or M 桅. 2) the overexpression of HPV16E2 and its TAD up-regulated the secretion of TNF- 伪 and IL-1 尾 by M 桅. High expression of HPV16E2 and TAD could induce apoptosis of M 桅.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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