GM-CSF和BZLF1融合基因重组腺病毒表达载体的构建

发布时间：2018-01-15 06:18

本文关键词：GM-CSF和BZLF1融合基因重组腺病毒表达载体的构建　出处：《青岛大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的将人粒细胞-巨噬细胞集落刺激因子(GM-CSF)编码基因与EB病毒(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,旨在发挥融合基因编码产物GM-SCF增强抗肿瘤免疫,以及BZLF1诱导潜伏期EBV进入裂解期复制的作用,为协同杀伤EBV阳性肿瘤细胞提供实验基础和理论依据。方法逆转录-聚合酶链反应分别获得目的基因GM-CSF和BZLF1编码序列的cDNA,采用剪接式重叠延伸(spliced overlap extension, SOE)技术将两段基因通过多肽接头(Gly4Ser) 3的DNA编码序列进行连接,构建融合基因BZGM。将融合基因BZGM定向亚克隆至pAdTrack-CMV穿梭质粒,在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因BZGM真核表达载体pAd-BZGM。经抗生素培养板筛选重组体,然后转染293细胞,获得复制缺陷型重组腺病毒vAd-BZGM。RT-PCR和Western blotting鉴定重组腺病毒感染人鼻咽癌CNE细胞的情况；MTT法检测腺病毒感染CNE后细胞的增殖情况。结果①将目的基因GM-CSF和BZLF1构建成融合基因BZGM。②构建融合基因BZGM重组腺病毒表达载体pAd-BZGM,测序鉴定结果表明序列正确无误。③将真核表达载体pAd-BZGM转染293细胞,包装获得重组腺病毒vAd-BZGM。④Western blotting和RT-PCR结果显示重组腺病毒感染的CNE细胞可检测到融合基因和EBV早期基因BMRF1表达,表明该融合基因能够诱导病毒从潜伏期进入裂解增殖期。⑤MTT法检测显示重组腺病毒感染CNE细胞后抑制细胞增殖的作用与所设对照组比较差异具有统计学意义(P0.05) 结论成功构建了GM-CSF和BZLF1融合基因BZGM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-BZGM,重组腺病毒vAd-BZGM可在靶细胞内稳定表达目的基因,可有效诱导潜伏状态EBV进入裂解期复制,进而抑制细胞的增殖,为进一步探讨BZGM的功能奠定了实验基础。
[Abstract]:Objective to fuse the human granulocyte-macrophage colony-stimulating factor (GM-CSF) coding gene with EBV immediate early gene (BZLF1). The recombinant adenovirus expression vector of the fusion gene was constructed in order to play the role of GM-SCF, the encoding product of the fusion gene, in enhancing anti-tumor immunity and in inducing the replication of latent EBV into lytic phase by BZLF1. To provide experimental and theoretical basis for synergistic killing of EBV positive tumor cells. Methods reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain the cDNA encoding the target gene GM-CSF and BZLF1 respectively. Spliced overlap extension. Soe technique linked the two segments of the gene through the DNA coding sequence of the polypeptide junction Gly4Ser3. The fusion gene BZGM was subcloned into pAdTrack-CMV shuttle plasmid. The homologous recombination of shuttle plasmid and skeleton plasmid pAdEasy-1 was completed in E. coli BJ5183. The fusion gene BZGM eukaryotic expression vector pAd-BZGMwas constructed. The recombinant was screened by antibiotic culture plate and then transfected into 293 cells. Replication-deficient recombinant adenovirus vAd-BZGM.RT-PCR and Western blotting were obtained to identify the infection of human nasopharyngeal carcinoma (NPC) CNE cells by recombinant adenovirus. The proliferation of adenovirus infected with CNE was detected by MTT. Results 1 the target gene GM-CSF and BZLF1 were constructed into the fusion gene BZGM.2 to construct the recombinant adenovirus expression vector pAd-BZGM of the fusion gene BZGM. The sequencing results showed that the sequence was correct. 3. The eukaryotic expression vector pAd-BZGM was transfected into 293 cells. Recombinant adenovirus vAd-BZGM.4Western obtained by packaging. The results of blotting and RT-PCR showed that the expression of fusion gene and early EBV gene BMRF1 could be detected in CNE cells infected by recombinant adenovirus. The results showed that the fusion gene could induce the virus from incubation period to lytic proliferation phase. 5 MTT assay showed that the inhibitory effect of recombinant adenovirus on cell proliferation after infection with CNE cells was statistically different from that of the control group. Scientific meaning (. P0.05) Conclusion the recombinant adenovirus expression vector of GM-CSF and BZLF1 fusion gene BZGM was successfully constructed and the recombinant adenovirus vAd-BZGM was obtained by packaging in 293 cells. Recombinant adenovirus vAd-BZGM can stably express the target gene in the target cells, and can effectively induce latent EBV into the lytic phase of replication, and then inhibit the proliferation of cells. It lays the experimental foundation for further discussing the function of BZGM.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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