人乳头瘤病毒18型E2蛋白及其删除突变体对Mφ分泌与凋亡的影响

发布时间：2018-01-18 09:28

本文关键词：人乳头瘤病毒18型E2蛋白及其删除突变体对Mφ分泌与凋亡的影响　出处：《南华大学》2008年硕士论文　论文类型：学位论文

更多相关文章： HPV 18 E2蛋白 MΦ 凋亡 细胞因子

【摘要】： 目的:构建人乳头瘤病毒18型(HPV 18)E2蛋白N端结构域(TAD)和C端结构域(DBD)与绿色荧光蛋白(EGFP)融合蛋白真核表达载体pEGFP-C1/TAD和pEGFP-C1/DBD,将其与pEGFP-C1/E2及pEGFP-C1分别转染THP-1巨噬细胞(macrophage, MΦ),分析各组培养基上清中TNF-α和IL-1β的含量以及MΦ凋亡率,为进一步研究E2蛋白在HPV 18致癌机制中的作用提供一定的实验依据。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E2蛋白N端和C端基因。将PCR产物纯化后与pEGFP-C1载体连接,转化E.coli JM109并提取质粒,经酶切和测序鉴定,筛选阳性克隆。 (2)将质粒pEGFP-C1/TAD、pEGFP-C1/DBD、pEGFP-C1/E2、pEGFP-C1分别为4组,即GFP-TAD组、GFP-DBD组、GFP-E2组、GFP组;每组质粒0.5μg/ml分别转染MΦ,并设空白对照组(Control)。转染48 h后,利用Western-blot和荧光显微镜检测它们的表达与定位。 (3) ELISA检测各组细胞培养基上清中TNF-α和IL-1β的含量,RT-PCR检测其mRNA表达;收集MΦ,利用染色法和流式细胞术(FCM)观察检测MΦ凋亡。通过统计软件SPSS13.0对实验数据进行统计分析。结果: (1) PCR产物连接至pEGFP-C1载体上后,双酶切及测序结果表明目的片段与HPV18 E2基因TAD、DBD序列完全一致。所构建的真核表达载体pEGFP-C1/TAD、pEGFP-C1/DBD以及pEGFP-C1/E2、pEGFP-C1分别转染THP-1 MΦ48 h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胞浆,GFP在细胞核浆内呈均匀表达。Western-blot结果显示,在THP-1 MΦ中表达分子量分别约为29.4 kD、53.4 kD、70.7 kD、38.6 kD的条带,与预期GFP、GFP-TAD、GFP-E2、GFP-DBD大小一致。 (2)转染THP-1 MΦ48 h后,GFP-E2、GFP-TAD组培养上清中TNF-α、IL-1β的含量明显高于Control组(P0.001),且两组之间TNF-α浓度存在统计学差异(P0.05);GFP-TAD组IL-1β含量略高于GFP- E2组,但差异无显著性(P0.05);而GFP- DBD、GFP组TNF-α及IL-1β含量与对照组比较,无统计学差异(P0.05)。TNF-α和IL-1βmRNA的表达量与上清液中TNF-α和IL-1β含量一致。 (3) GFP-E2、GFP-TAD组MΦ凋亡率明显高于Control组(P0.001),且GFP-TAD组MΦ凋亡率高于GFP-E2组(P0.05);但GFP-DBD、GFP组与Control组比较,MΦ凋亡率无统计学差异(P0.05)。结论: (1)成功地扩增了HPV18 E2TAD和DBD基因,构建了真核表达载体pEGFP-C1/TAD、pEGFP-C1/DBD,且其与质粒pEGFP-C1/E2和pEGFP-C1均能在THP-1 MΦ中表达,GFP-DBD融合蛋白定位于细胞核内,GFP-TAD定位于细胞浆。 (2) HPV18 E2及其TAD与GFP融合蛋白瞬时高表达上调THP-1 MΦ分泌TNF-α和IL-1β。 (3) HPV18 E2及其TAD与GFP融合蛋白瞬时高表达诱导THP-1 MΦ凋亡。
[Abstract]:Objective: to construct human papillomavirus type 18 (HPV18) E2 protein N-terminal domain (TAD) and C-terminal domain (DBD) and green fluorescent protein (EGFP). The eukaryotic expression vector of fusion protein pEGFP-C1/TAD and pEGFP-C1/DBD. It was transfected into THP-1 macrophage (M 桅) with pEGFP-C1/E2 and pEGFP-C1, respectively. The contents of TNF- 伪 and IL-1 尾 and the apoptosis rate of M 桅 in the supernatant of each group were analyzed, which provided a certain experimental basis for the further study of the role of E2 protein in the carcinogenesis of HPV 18. Methods: Primer5.0 primer design software was used to design primers. The N-terminal and C-terminal genes of E2 protein were amplified by polymerase chain reaction (PCR). The PCR product was purified and ligated with pEGFP-C1 vector to transform into E. coli JM109 and extract the plasmid. The positive clones were screened by enzyme digestion and sequencing. (2) the plasmid pEGFP-C1 / TAD1 / DBDN pEGFP-C1 / E2pEGFP-C1 was divided into 4 groups, namely, GFP-TAD group. GFP-DBD group: GFP-E2 group, GFP group; Each group of plasmids 0.5 渭 g / ml was transfected with M 桅, and a blank control group was set up for 48 h after transfection. Western-blot and fluorescence microscope were used to detect their expression and localization. ELISA was used to detect the content of TNF- 伪 and IL-1 尾 in the supernatant of cell culture medium. RT-PCR was used to detect the mRNA expression of TNF- 伪 and IL-1 尾. The apoptosis of M 桅 was observed by staining and flow cytometry, and the experimental data were analyzed by statistical software SPSS13.0. Results: 1) after the PCR product was ligated to the pEGFP-C1 vector, the result of double enzyme digestion and sequencing showed that the target fragment and HPV18 E2 gene TAD. The constructed eukaryotic expression vector pEGFP-C1 / TAD1 / DBD pEGFP-C1 / DBD and pEGFP-C1/E2. PEGFP-C1 transfected THP-1 M 桅 48 h later, the GFP-DBD fusion protein was located in the nucleus of GFP-TAD and located in the cytoplasm of GFP-E2. GFP was expressed in the nucleus and cytoplasm, but the fluorescence intensity of the GFP-E2 group was stronger than that of the cytoplasm. The expression of GFP was homogeneously expressed in cytoplasm. Western-blot showed that the molecular weight of GFP expressed in THP-1 M 桅 was about 29.4 KD and 53.4 KD, respectively. The band of 38.6 KD of 70.7 KD is the same as that of GFP-TAD2 GFP-DBD. (2) THP-1 M 桅 48 h after transfection, TNF- 伪 in the culture supernatant of GFP-E2 + GFP-TAD group. The content of IL-1 尾 was significantly higher than that of Control group (P 0.001), and the concentration of TNF- 伪 was significantly different between the two groups (P 0.05). The content of IL-1 尾 in GFP-TAD group was slightly higher than that in GFP-E2 group, but there was no significant difference (P 0.05). The contents of TNF- 伪 and IL-1 尾 in GFP group were compared with those in control group. There was no statistical difference between the expression of P0.05A. TNF- 伪 and IL-1 尾 mRNA and the content of TNF- 伪 and IL-1 尾 in supernatant. The apoptosis rate of M 桅 in GFP-E2GFP-TAD group was significantly higher than that in Control group (P 0.001). The apoptosis rate of M 桅 in GFP-TAD group was higher than that in GFP-E2 group (P 0.05). However, there was no significant difference in apoptosis rate of M 桅 between GFP-DBDFP group and Control group (P 0.05). Conclusion: (1) HPV18 E2TAD and DBD genes were amplified successfully, and the eukaryotic expression vector pEGFP-C1 / TAD-1 / pEGFP-C1 / DBD was constructed. The fusion protein was expressed in THP-1 M 桅 with plasmid pEGFP-C1/E2 and pEGFP-C1, and the fusion protein was located in the nucleus. GFP-TAD was located in the cytoplasm. 2) the transient overexpression of HPV18 E2 and its TAD / GFP fusion protein upregulated the secretion of TNF- 伪 and IL-1 尾 by THP-1 M 桅. Apoptosis of THP-1 M 桅 was induced by transient overexpression of HPV18 E2 and its TAD and GFP fusion protein.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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