携带白蛋白启动子和IDO基因的重组腺病毒载体的构建
发布时间：2018-10-22 15:19
【摘要】： 【目的】构建携带小鼠白蛋白启动子和IDO基因的重组腺病毒载体,研究其在肝脏Hepa 1-6细胞的转录水平及蛋白表达情况。 【方法】按照引物设计的基本原则设计白蛋白启动子引物,人工合成白蛋白启动子,亚克隆至没有启动子的腺病毒穿梭载体pAdTrack的XbaI/HindIII酶切位点,构建携带有小鼠白蛋白启动子的pAdTrack-PmALB载体;酶切含有小鼠全长IDOcDNA的PCOUS-2质粒,亚克隆至穿梭载体pAdTrack-PmALB、pAdTrack-CMV上的XhoI/EcoRV酶切位点,在BJ5183细菌中和AdEasy-1进行同源重组,生成并筛选含有目的基因的腺病毒骨架质粒pAdEasy-CMV/mIDO和pAdEasy-malb/mIDO的阳性克隆,酶切、测序鉴定正确后,脂质体法转染AD-293细胞进行包装,扩增,检测病毒滴度,RT-PCR和荧光显微镜鉴定和检测重组腺病毒转染AD-293细胞后IDO的表达。将含有目的基因mIDO的病毒转染Hepa 1-6细胞,RT-PCR和Western Blot法分别检测Hepa 1-6细胞中mIDO基因和蛋白表达。 【结果】经酶切及测序证实携带白蛋白启动子和IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,病毒感染滴度为2.9×106 pfu/ml,并且能够感染Hepa 1-6细胞,RT-PCR和Western Blot可以检测到IDO在Hepa 1-6细胞内表达。 【结论】成功构建了携带白蛋白启动子和小鼠IDO基因的重组腺病毒载体,能够感染Hepa 1-6细胞并在转录和蛋白水平表达,为探讨IDO的生物学功能奠定了基础。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
【相似文献】
中国期刊全文数据库 前10条
1 侯卫平;黄钢;翟志芳;袁发焕;;小鼠IDO基因重组腺病毒载体的构建表达和鉴定[J];重庆医学;2007年05期
2 谢启超;王玲俐;陈正堂;;IDO与肿瘤免疫逃逸[J];重庆医学;2007年13期
3 谢启超;王玲俐;朱波;王亚丽;顾健滕;陈正堂;;IDO基因转染小鼠Lewis肺癌细胞体外诱导Treg细胞增殖的研究[J];重庆医学;2007年21期
4 李玉品;黄建鸣;李戈;;急性髓系白血病细胞诱导免疫耐受及其机制的研究进展[J];实用医院临床杂志;2011年04期
5 布晓坤;李宏钊;邢冬红;黄焕军;白虹;;IFN-γ在沙眼衣原体呼吸道感染中免疫防御机制的探讨[J];中国免疫学杂志;2007年12期
6 刘瑶;林菊生;熊杰;谭锦泉;张强;常莹;任精华;;肝脏特异性表达人遗传印记基因PEG10转基因载体的构建和鉴定[J];胃肠病学和肝病学杂志;2008年12期
7 刘凤娟;董e，
本文编号：2287529
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2287529.html
最近更新
教材专著
