本文選題：LMP2A + BZLF1　； 參考：《青島大學(xué)》2006年碩士論文

【摘要】：目的 將EBV潛伏膜蛋白編碼基因LMP2A和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,旨在同時(shí)發(fā)揮LMP2A誘導(dǎo)特異性CTL和BZLF1基因產(chǎn)物誘導(dǎo)潛伏期EBV進(jìn)入裂解期復(fù)制的作用,從而協(xié)同殺傷EBV陽(yáng)性腫瘤細(xì)胞,為EBV相關(guān)腫瘤特異性治療的臨床應(yīng)用研究奠定試驗(yàn)基礎(chǔ)。 方法 自EBV陽(yáng)性細(xì)胞提取總RNA,經(jīng)逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)分別獲得LMP2A和BZLF1編碼序列的cDNA,采用剪接式重疊延伸(spliced overlap exetension,SOE)技術(shù),將兩段基因通過(guò)多肽接頭(Gly_4Ser)_3 DNA序列連接以獲得融合基因(Z2A)。進(jìn)一步將Z2A融合基因定向亞克隆到pAdTrack-CMV質(zhì)粒上,在原核細(xì)胞E.coli BJ5183中完成穿梭質(zhì)粒與骨架質(zhì)粒之間的高效同源重組,構(gòu)建Z2A真核表達(dá)載體pAd-Z2A。經(jīng)抗生素培養(yǎng)板篩選重組體,然后轉(zhuǎn)染293細(xì)胞,包裝獲得重組腺病毒vAd-Z2A。流式細(xì)胞術(shù)檢測(cè)重組腺病毒感染EBV陽(yáng)性鼻咽癌細(xì)胞CNE誘導(dǎo)的凋亡。 結(jié)果 ①重組腺病毒載體經(jīng)限制性核酸內(nèi)切酶酶切,電泳后可觀察到31kb和4.5kb兩條DNA條帶,測(cè)序鑒定結(jié)果表明序列正確;②RT-PCR顯示重組腺病毒感染的CNE細(xì)胞可檢測(cè)到融合基因表達(dá);③Western Blotting可檢測(cè)到融合蛋白的表達(dá);④流式細(xì)胞術(shù)檢測(cè)顯示重組腺病毒感染CNE細(xì)胞誘導(dǎo)的凋亡與vAd-LacZ載體對(duì)照和空白對(duì)照比較差異均有統(tǒng)計(jì)學(xué)意義(P0.001)。 結(jié)論 本研究成功構(gòu)建了EBV潛伏膜蛋白基因LMP2A和即刻早期基因BZLF1融合基因的重組腺病毒表達(dá)載體,并在293細(xì)胞中包裝獲得重組腺病毒vAd-Z2A,重組腺病毒vAd-Z2A可在靶細(xì)胞內(nèi)穩(wěn)定表達(dá)目的基因并能誘導(dǎo)EBV陽(yáng)性CNE細(xì)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.
【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】：2006
【分類號(hào)】：R346

【共引文獻(xiàn)】

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