【摘要】： 【目的】構(gòu)建攜帶小鼠白蛋白啟動子和IDO基因的重組腺病毒載體,研究其在肝臟Hepa 1-6細(xì)胞的轉(zhuǎn)錄水平及蛋白表達(dá)情況。 【方法】按照引物設(shè)計的基本原則設(shè)計白蛋白啟動子引物,人工合成白蛋白啟動子,亞克隆至沒有啟動子的腺病毒穿梭載體pAdTrack的XbaI/HindIII酶切位點,構(gòu)建攜帶有小鼠白蛋白啟動子的pAdTrack-PmALB載體;酶切含有小鼠全長IDOcDNA的PCOUS-2質(zhì)粒,亞克隆至穿梭載體pAdTrack-PmALB、pAdTrack-CMV上的XhoI/EcoRV酶切位點,在BJ5183細(xì)菌中和AdEasy-1進(jìn)行同源重組,生成并篩選含有目的基因的腺病毒骨架質(zhì)粒pAdEasy-CMV/mIDO和pAdEasy-malb/mIDO的陽性克隆,酶切、測序鑒定正確后,脂質(zhì)體法轉(zhuǎn)染AD-293細(xì)胞進(jìn)行包裝,擴(kuò)增,檢測病毒滴度,RT-PCR和熒光顯微鏡鑒定和檢測重組腺病毒轉(zhuǎn)染AD-293細(xì)胞后IDO的表達(dá)。將含有目的基因mIDO的病毒轉(zhuǎn)染Hepa 1-6細(xì)胞,RT-PCR和Western Blot法分別檢測Hepa 1-6細(xì)胞中mIDO基因和蛋白表達(dá)。 【結(jié)果】經(jīng)酶切及測序證實攜帶白蛋白啟動子和IDO基因重組腺病毒載體構(gòu)建成功,RT-PCR檢測到轉(zhuǎn)染后AD-293細(xì)胞內(nèi)IDO的表達(dá),病毒感染滴度為2.9×106 pfu/ml,并且能夠感染Hepa 1-6細(xì)胞,RT-PCR和Western Blot可以檢測到IDO在Hepa 1-6細(xì)胞內(nèi)表達(dá)。 【結(jié)論】成功構(gòu)建了攜帶白蛋白啟動子和小鼠IDO基因的重組腺病毒載體,能夠感染Hepa 1-6細(xì)胞并在轉(zhuǎn)錄和蛋白水平表達(dá),為探討IDO的生物學(xué)功能奠定了基礎(chǔ)。
[Abstract]:[objective] to construct a recombinant adenovirus vector carrying mouse albumin promoter and IDO gene. To study the transcriptional level and protein expression of Hepa 1-6 cells in liver. [methods] Albumin promoter primer was designed according to the basic principle of primer design, and albumin promoter was synthesized. Subcloned to the XbaI/HindIII site of the adenovirus shuttle vector pAdTrack without promoter, the pAdTrack-PmALB vector containing mouse albumin promoter was constructed, and the PCOUS-2 plasmid containing mouse full-length IDOcDNA was subcloned to the XhoI/EcoRV restriction site on the pAdTrack-PmALB,pAdTrack-CMV shuttle vector. After homologous recombination of BJ5183 bacteria and AdEasy-1, the positive clones of adenovirus skeleton plasmids pAdEasy-CMV/mIDO and pAdEasy-malb/mIDO containing the target gene were generated and screened. After restriction endonuclease digestion and sequencing, the AD-293 cells were transfected with liposome for packaging and amplification. RT-PCR and fluorescence microscopy were used to identify and detect the expression of IDO after transfection of recombinant adenovirus into AD-293 cells. The virus containing the target gene mIDO was transfected into Hepa 1-6 cells. The expression of mIDO gene and protein in Hepa 1-6 cells were detected by RT-PCR and Western Blot respectively. [results] it was confirmed by restriction endonuclease digestion and sequencing to carry albumin promoter and IDO. The recombinant adenovirus vector was successfully constructed and the expression of IDO in AD-293 cells was detected by RT-PCR. The viral titer is 2.9 脳 10 ~ 6 pfu/ml, and can infect Hepa 1-6 cells. The expression of IDO in Hepa 1-6 cells can be detected by RT-PCR and Western Blot. [conclusion] the recombinant plasmid carrying albumin promoter and mouse IDO gene was successfully constructed. Recombinant adenovirus vector, It can infect Hepa 1-6 cells and express at the level of transcription and protein, which lays a foundation for studying the biological function of IDO.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.4

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