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  本文關(guān)鍵詞：GM-CSF和BZLF1融合基因重組腺病毒表達(dá)載體的構(gòu)建　出處：《青島大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

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【摘要】： 目的將人粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)編碼基因與EB病毒(EBV)即刻早期基因(BZLF1)進(jìn)行融合,構(gòu)建融合基因的重組腺病毒表達(dá)載體,旨在發(fā)揮融合基因編碼產(chǎn)物GM-SCF增強(qiáng)抗腫瘤免疫,以及BZLF1誘導(dǎo)潛伏期EBV進(jìn)入裂解期復(fù)制的作用,為協(xié)同殺傷EBV陽(yáng)性腫瘤細(xì)胞提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 方法逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)分別獲得目的基因GM-CSF和BZLF1編碼序列的cDNA,采用剪接式重疊延伸(spliced overlap extension, SOE)技術(shù)將兩段基因通過(guò)多肽接頭(Gly4Ser) 3的DNA編碼序列進(jìn)行連接,構(gòu)建融合基因BZGM。將融合基因BZGM定向亞克隆至pAdTrack-CMV穿梭質(zhì)粒,在原核細(xì)胞E.coli BJ5183中完成穿梭質(zhì)粒與骨架質(zhì)粒pAdEasy-1的同源重組,構(gòu)建融合基因BZGM真核表達(dá)載體pAd-BZGM。經(jīng)抗生素培養(yǎng)板篩選重組體,然后轉(zhuǎn)染293細(xì)胞,獲得復(fù)制缺陷型重組腺病毒vAd-BZGM。RT-PCR和Western blotting鑒定重組腺病毒感染人鼻咽癌CNE細(xì)胞的情況；MTT法檢測(cè)腺病毒感染CNE后細(xì)胞的增殖情況。 結(jié)果①將目的基因GM-CSF和BZLF1構(gòu)建成融合基因BZGM。②構(gòu)建融合基因BZGM重組腺病毒表達(dá)載體pAd-BZGM,測(cè)序鑒定結(jié)果表明序列正確無(wú)誤。③將真核表達(dá)載體pAd-BZGM轉(zhuǎn)染293細(xì)胞,包裝獲得重組腺病毒vAd-BZGM。④Western blotting和RT-PCR結(jié)果顯示重組腺病毒感染的CNE細(xì)胞可檢測(cè)到融合基因和EBV早期基因BMRF1表達(dá),表明該融合基因能夠誘導(dǎo)病毒從潛伏期進(jìn)入裂解增殖期。⑤MTT法檢測(cè)顯示重組腺病毒感染CNE細(xì)胞后抑制細(xì)胞增殖的作用與所設(shè)對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05) 結(jié)論成功構(gòu)建了GM-CSF和BZLF1融合基因BZGM重組腺病毒表達(dá)載體,并在293細(xì)胞中包裝獲得重組腺病毒vAd-BZGM,重組腺病毒vAd-BZGM可在靶細(xì)胞內(nèi)穩(wěn)定表達(dá)目的基因,可有效誘導(dǎo)潛伏狀態(tài)EBV進(jìn)入裂解期復(fù)制,進(jìn)而抑制細(xì)胞的增殖,為進(jìn)一步探討B(tài)ZGM的功能奠定了實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373

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相關(guān)期刊論文 前1條

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本文編號(hào)：1427144