發(fā)布時(shí)間：2018-01-18 09:28

  本文關(guān)鍵詞：人乳頭瘤病毒18型E2蛋白及其刪除突變體對(duì)Mφ分泌與凋亡的影響　出處：《南華大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HPV 18 E2蛋白 MΦ 凋亡 細(xì)胞因子

【摘要】： 目的:構(gòu)建人乳頭瘤病毒18型(HPV 18)E2蛋白N端結(jié)構(gòu)域(TAD)和C端結(jié)構(gòu)域(DBD)與綠色熒光蛋白(EGFP)融合蛋白真核表達(dá)載體pEGFP-C1/TAD和pEGFP-C1/DBD,將其與pEGFP-C1/E2及pEGFP-C1分別轉(zhuǎn)染THP-1巨噬細(xì)胞(macrophage, MΦ),分析各組培養(yǎng)基上清中TNF-α和IL-1β的含量以及MΦ凋亡率,為進(jìn)一步研究E2蛋白在HPV 18致癌機(jī)制中的作用提供一定的實(shí)驗(yàn)依據(jù)。 方法: (1)用Primer5.0引物設(shè)計(jì)軟件設(shè)計(jì)引物,聚合酶鏈反應(yīng)(PCR)擴(kuò)增E2蛋白N端和C端基因。將PCR產(chǎn)物純化后與pEGFP-C1載體連接,轉(zhuǎn)化E.coli JM109并提取質(zhì)粒,經(jīng)酶切和測(cè)序鑒定,篩選陽(yáng)性克隆。 (2)將質(zhì)粒pEGFP-C1/TAD、pEGFP-C1/DBD、pEGFP-C1/E2、pEGFP-C1分別為4組,即GFP-TAD組、GFP-DBD組、GFP-E2組、GFP組;每組質(zhì)粒0.5μg/ml分別轉(zhuǎn)染MΦ,并設(shè)空白對(duì)照組(Control)。轉(zhuǎn)染48 h后,利用Western-blot和熒光顯微鏡檢測(cè)它們的表達(dá)與定位。 (3) ELISA檢測(cè)各組細(xì)胞培養(yǎng)基上清中TNF-α和IL-1β的含量,RT-PCR檢測(cè)其mRNA表達(dá);收集MΦ,利用染色法和流式細(xì)胞術(shù)(FCM)觀察檢測(cè)MΦ凋亡。通過(guò)統(tǒng)計(jì)軟件SPSS13.0對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果: (1) PCR產(chǎn)物連接至pEGFP-C1載體上后,雙酶切及測(cè)序結(jié)果表明目的片段與HPV18 E2基因TAD、DBD序列完全一致。所構(gòu)建的真核表達(dá)載體pEGFP-C1/TAD、pEGFP-C1/DBD以及pEGFP-C1/E2、pEGFP-C1分別轉(zhuǎn)染THP-1 MΦ48 h后,GFP-DBD融合蛋白定位于細(xì)胞核內(nèi),GFP-TAD定位于細(xì)胞漿,GFP-E2、GFP在細(xì)胞核、漿內(nèi)均有表達(dá),但GFP-E2表達(dá)組細(xì)胞核熒光亮度強(qiáng)于細(xì)胞胞漿,GFP在細(xì)胞核漿內(nèi)呈均勻表達(dá)。Western-blot結(jié)果顯示,在THP-1 MΦ中表達(dá)分子量分別約為29.4 kD、53.4 kD、70.7 kD、38.6 kD的條帶,與預(yù)期GFP、GFP-TAD、GFP-E2、GFP-DBD大小一致。 (2)轉(zhuǎn)染THP-1 MΦ48 h后,GFP-E2、GFP-TAD組培養(yǎng)上清中TNF-α、IL-1β的含量明顯高于Control組(P0.001),且兩組之間TNF-α濃度存在統(tǒng)計(jì)學(xué)差異(P0.05);GFP-TAD組IL-1β含量略高于GFP- E2組,但差異無(wú)顯著性(P0.05);而GFP- DBD、GFP組TNF-α及IL-1β含量與對(duì)照組比較,無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。TNF-α和IL-1βmRNA的表達(dá)量與上清液中TNF-α和IL-1β含量一致。 (3) GFP-E2、GFP-TAD組MΦ凋亡率明顯高于Control組(P0.001),且GFP-TAD組MΦ凋亡率高于GFP-E2組(P0.05);但GFP-DBD、GFP組與Control組比較,MΦ凋亡率無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論: (1)成功地?cái)U(kuò)增了HPV18 E2TAD和DBD基因,構(gòu)建了真核表達(dá)載體pEGFP-C1/TAD、pEGFP-C1/DBD,且其與質(zhì)粒pEGFP-C1/E2和pEGFP-C1均能在THP-1 MΦ中表達(dá),GFP-DBD融合蛋白定位于細(xì)胞核內(nèi),GFP-TAD定位于細(xì)胞漿。 (2) HPV18 E2及其TAD與GFP融合蛋白瞬時(shí)高表達(dá)上調(diào)THP-1 MΦ分泌TNF-α和IL-1β。 (3) HPV18 E2及其TAD與GFP融合蛋白瞬時(shí)高表達(dá)誘導(dǎo)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- 偽 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- 偽 and IL-1 尾 in the supernatant of cell culture medium. RT-PCR was used to detect the mRNA expression of TNF- 偽 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- 偽 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- 偽 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- 偽 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- 偽 and IL-1 尾 mRNA and the content of TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373

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本文編號(hào)：1440359