LMP2A和BZLF1融合基因重组腺病毒表达载体的构建和初步应用

发布时间：2018-06-25 15:56

本文选题：LMP2A + BZLF1　；参考：《青岛大学》2006年硕士论文

【摘要】：目的将EBV潜伏膜蛋白编码基因LMP2A和EBV即刻早期基因BZLF1进行融合,构建融合基因的重组腺病毒表达载体,旨在同时发挥LMP2A诱导特异性CTL和BZLF1基因产物诱导潜伏期EBV进入裂解期复制的作用,从而协同杀伤EBV阳性肿瘤细胞,为EBV相关肿瘤特异性治疗的临床应用研究奠定试验基础。方法自EBV阳性细胞提取总RNA,经逆转录-聚合酶链反应分别获得LMP2A和BZLF1编码序列的cDNA,采用剪接式重叠延伸(spliced overlap exetension,SOE)技术,将两段基因通过多肽接头(Gly_4Ser)_3 DNA序列连接以获得融合基因(Z2A)。进一步将Z2A融合基因定向亚克隆到pAdTrack-CMV质粒上,在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒之间的高效同源重组,构建Z2A真核表达载体pAd-Z2A。经抗生素培养板筛选重组体,然后转染293细胞,包装获得重组腺病毒vAd-Z2A。流式细胞术检测重组腺病毒感染EBV阳性鼻咽癌细胞CNE诱导的凋亡。结果 ①重组腺病毒载体经限制性核酸内切酶酶切,电泳后可观察到31kb和4.5kb两条DNA条带,测序鉴定结果表明序列正确;②RT-PCR显示重组腺病毒感染的CNE细胞可检测到融合基因表达;③Western Blotting可检测到融合蛋白的表达;④流式细胞术检测显示重组腺病毒感染CNE细胞诱导的凋亡与vAd-LacZ载体对照和空白对照比较差异均有统计学意义(P0.001)。结论本研究成功构建了EBV潜伏膜蛋白基因LMP2A和即刻早期基因BZLF1融合基因的重组腺病毒表达载体,并在293细胞中包装获得重组腺病毒vAd-Z2A,重组腺病毒vAd-Z2A可在靶细胞内稳定表达目的基因并能诱导EBV阳性CNE细胞的凋亡。
[Abstract]:Objective to construct the recombinant adenovirus expression vector of EBV latent membrane protein (EBV) encoding gene LMP2A and EBV immediate early gene BZLF1. The aim of this study was to exert the effect of LMP2A inducing specific CTL and BZLF1 gene product induction latent period EBV into lytic phase and to kill EBV positive tumor cells simultaneously, and to lay the experimental foundation for the clinical application of EBV related tumor specific therapy. Methods Total RNAs were extracted from EBV-positive cells. The cDNA of LMP2A and BZLF1 coding sequences were obtained by reverse transcription-polymerase chain reaction (RT-PCR). Splicing overlapping extension (spliced overlap extension (SOE) technique was used. The fusion gene (Z2A) was obtained by ligating the two segments of the gene through the peptide junction (GlyS4Ser) into the tip3 DNA sequence. Furthermore, Z2A fusion gene was subcloned into pAdTrack-CMV plasmid, and the shuttle plasmid and skeleton plasmid were recombined into E. coli BJ5183 to construct Z2A eukaryotic expression vector pAd-Z2A. Recombinant adenovirus vAd-Z2A was obtained by screening recombinant adenovirus by antibiotic culture plate, then transfected into 293 cells. Apoptosis induced by EBV positive nasopharyngeal carcinoma cell line CNE was detected by flow cytometry. Results (1) the recombinant adenovirus vector was digested by restriction endonuclease and the 31kb and 4.5kb bands were observed by electrophoresis. The sequencing results showed that the sequence was correct. 2RT-PCR showed that fusion gene expression could be detected in CNE cells infected with recombinant adenovirus. Western blotting could detect the expression of fusion protein. 4 the results of flow cytometry showed that the apoptosis induced by recombinant adenovirus infected CNE cells was significantly different from that of vAd-LacZ vector and blank control (P0.001). Conclusion the recombinant adenovirus expression vector of EBV latent membrane protein gene LMP2A and immediate early gene BZLF1 fusion gene was successfully constructed. The recombinant adenovirus vAd-Z2A was packaged in 293 cells. The recombinant adenovirus vAd-Z2A could stably express the target gene and induce the apoptosis of EBV-positive CNE cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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