發(fā)布時間：2018-03-03 17:44

  本文選題：EB病毒　切入點：重組腺病毒　出處：《青島大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的構建EBV潛伏期膜蛋白LMP2A編碼基因的腺病毒表達載體，初步探討LMP2A表達對靶細胞SGC-7901的生物學作用。 方法RT-PCR擴增獲取目的基因LMP2A，采用AdEasy系統(tǒng)構建攜帶目的基因的重組腺病毒載體pAd-2A，脂質體法將重組質粒pAd-2A轉染HEK293細胞包裝重組腺病毒。選擇EBV陰性胃癌細胞SGC-7901作為靶細胞，試驗分重組腺病毒SGC-2A組，病毒對照SGC-GFP組和細胞對照SGC組3組，采用MTT實驗、透射電鏡觀察、流式細胞分析、激光共聚焦等技術檢測目的基因LMP2A表達對靶細胞生物學行為的影響。 結果①限制性酶切、PCR及測序鑒定證實，目的基因LMP2A正確插入重組質粒，，HEK293細胞成功包裝出具有穩(wěn)定感染性的重組腺病毒，命名為vAd-2A，經測定病毒滴度為3.16×10~(12)pfu／L。②MTT檢測結果顯示：與SGC-GFP組和SGC組比較，SGC-2A組細胞從接種后第2天起表現(xiàn)出更強的增殖能力。③透射電鏡觀察SGC-2A組細胞的超微結構無明顯改變，而SGC-GFP組和SGC組則出現(xiàn)一系列變化，并可觀察到凋亡小體。④激光共聚焦分析表明，SGC-2A組細胞幾乎均可觀察到較強的cyclinE熒光信號；而SGC-GFP組僅部分細胞可觀察到cyclinE熒光信號，且較弱；未感染病毒的SGC組細胞則僅有少量細胞觀察到熒光。⑤流式細胞分析感染vAd-2A后48h和72h的SGC-7901細胞S期的比例明顯高于SGC-GFP和SGC兩對照組。 結論成功構建了EBV潛伏期膜蛋白基因LMP2A的重組腺病毒表達載體，并在HEK293細胞中成功包裝出重組腺病毒；同時初步證實LMP2A可促進SGC-7901細胞增殖并抑制其凋亡，該作用可能與LMP2A誘導靶細胞cyclinE的表達有關。
[Abstract]:Objective to construct the adenovirus expression vector of EBV latent membrane protein (LMP2A) encoding gene and to explore the biological effect of LMP2A expression on target cell SGC-7901. Methods the target gene LMP2A was amplified by RT-PCR, and the recombinant adenovirus vector pAd-2A was constructed by AdEasy system. The recombinant plasmid pAd-2A was transfected into HEK293 cells to package the recombinant adenovirus by liposome method. The EBV negative gastric cancer cell SGC-7901 was selected as the target cell. The experiment was divided into three groups: recombinant adenovirus SGC-2A group, virus control SGC-GFP group and cell control SGC group. The effects of LMP2A expression on the biological behavior of target cells were detected by MTT experiment, transmission electron microscopy, flow cytometry and laser confocal focusing. Results 1 restriction endonuclease PCR and sequencing confirmed that the recombinant adenovirus with stable infection was successfully packaged by inserting LMP2A into the recombinant plasmid pHEK293. VAd-2A was named as vAd-2A. The results of MTT assay showed that compared with SGC-GFP group and SGC group, SGC-2A cells showed stronger proliferative ability from the second day after inoculation. 3 transmission electron microscope showed no obvious change in ultrastructure of SGC-2A group. In SGC-GFP group and SGC group, a series of changes were observed, and the apoptosis corpuscles were observed by confocal laser analysis. The results showed that cyclinE fluorescence signal was almost observed in SGC-2A group, while cyclinE fluorescence signal was observed in only a few cells in SGC-GFP group. Only a few of the cells in the SGC group without infection observed that the S phase ratio of SGC-7901 cells at 48h and 72h after vAd-2A infection was significantly higher than that in the SGC-GFP and SGC control groups. Conclusion the recombinant adenovirus expression vector of EBV latent membrane protein gene LMP2A was successfully constructed, and the recombinant adenovirus was successfully packaged in HEK293 cells, and it was preliminarily proved that LMP2A could promote the proliferation and inhibit the apoptosis of SGC-7901 cells. This effect may be related to the expression of cyclinE in target cells induced by LMP2A.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R735.2;R373

【參考文獻】

相關期刊論文 前4條

1 車向明,徐威,帆北修一,愛甲孝;Cyclin E及p53的表達與早期胃癌進展及淋巴結轉移的關系[J];西安醫(yī)科大學學報;2001年05期

2 施明;王福生;高蘭興;;腺病毒載體的研究進展[J];世界華人消化雜志;2000年11期

3 夏加增,王心南,楊繼昌,尹浩然,朱正鋼;細胞周期蛋白E在胃癌中的表達及其與細胞增殖活性的關系[J];江蘇臨床醫(yī)學雜志;1999年02期

4 Maria D Begnami;Andre L Montagnini;Andre L Vettore;Sueli Nonogaki;Mariana Brait;Alex Y Simoes-Sato;Andrea Q A Seixas;Fernando A Soares;;Differential expression of apoptosis related proteins and nitric oxide synthases in Epstein Barr associated gastric carcinomas[J];World Journal of Gastroenterology;2006年31期

本文編號：1562044