高表达hRAMP1基因腺病毒载体的构建及其在兔MSCs中表达的实验研究

发布时间：2018-05-23 08:14

  本文选题：腺病毒载体 + 骨髓间充质干细胞　； 参考：《遵义医学院》2010年硕士论文

【摘要】： 目的：构建重组腺病毒载体pAdxsi-EGFP-hRAMP1,转染兔骨髓间充质干细胞(BMSCs),观察其转染率及hRAMP1的mRNA及蛋白表达。方法：质粒pOTB7-hRAMP1用EcoRI+XhoI双酶切后回收hRAMP1片段,亚克隆至pShuttle-EGFP-CMV中,得到重组穿梭质粒；I-CeuI+I-Sce1双酶切处理pShuttle-EGFP-CMV-hRAMP1,回收EGFP-CMV-EGFP-hRAMP 1片段,亚克隆至腺病毒骨架载体pAdxsi,得到重组腺病毒质粒；重组腺病毒质粒酶切线性化后应用脂质体法转染293细胞,观察细胞出毒迹象并进行包装、收毒、冻融、扩增、再收毒并作毒种鉴定后以离心,透析方法纯化腺病毒进行滴度测定及检测。提取新鲜骨髓,体外分离、培养并鉴定MSCs。将带有hRAMP1和EGFP基因的腺病毒载体,感染第3代MSCs,荧光倒置显微镜下计算感染率。收集病毒感染后的MSCs行RT-PCR检测表明存在hRAMP1基因的mRNA表达,WesternBlot亦证实hRAMP1基因的蛋白表达。结果：构建的重组穿梭质pShuttle-EGFP-CMV-hRAMP1用EcoRI+XhoI双酶切,得到大小为0.8kbp (hRAMP1)和5.1kbp (pShuttle-EGFP-CMV)两个片段；重组腺病毒质粒pAdxsi-EGFP-CMV-hRAMP 1用XhoI酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段；重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用；重组腺病毒Ad-EGFP-hRAMP1 PCR鉴定可见164bp的阳性扩增条带；经多次重复感染后,病毒滴度检测达2.5×1011PFU/ml.成功转染Ad-EGFP-hRAMP1的兔MSCs可表达绿色荧光,当MOI值为800,转染效率为80%。RT-PCR检测转染后的MSCs有hRAMP1基因mRNA表达;Wester-blot检测转染后的MSCs有bRAMP1蛋白表达。结论：(1)成功构建了携带增强型绿色荧光蛋白基因和hRAMP1基因的重组腺病毒载体；(2)MSCs是一种理想的基因载体细胞,可用于hRAMP1的基因治疗。
[Abstract]:Aim: to construct recombinant adenovirus vector pAdxsi-EGFP-hRAMP1 and transfect it into rabbit bone marrow mesenchymal stem cells (BMSCs) and observe its transfection rate, mRNA and protein expression of hRAMP1. Methods: the recombinant shuttle plasmid pShuttle-EGFP-CMV-hRAMP1 was obtained by double enzyme digestion of plasmid pOTB7-hRAMP1, which was digested with EcoRI XhoI and subcloned into pShuttle-EGFP-CMV. The recombinant adenovirus plasmid was subcloned into adenovirus skeleton vector pAdxsi. the recombinant adenovirus plasmid was obtained by double digestion of pShuttle-EGFP-CMV-hRAMP1. The recombinant adenovirus plasmid was linearized by enzyme digestion and transfected into 293 cells by liposome method. The virulence of the cells was observed and packaged, poisoned, freeze-thawed, amplified, then poisoned and identified by centrifugation. The titer of adenovirus purified by dialysis was determined and detected. Fresh bone marrow was extracted, isolated, cultured and identified in vitro. The adenovirus vector containing hRAMP1 and EGFP genes was infected with MSCs of the third generation and the infection rate was calculated by fluorescence inverted microscope. The mRNA expression of hRAMP1 gene was detected by RT-PCR analysis after MSCs infection. Western Blot also confirmed the protein expression of hRAMP1 gene. Results: the recombinant shuttle pShuttle-EGFP-CMV-hRAMP1 was digested with EcoRI XhoI to obtain two fragments, 0.8kbp hRAMP1) and 5.1kbp pShuttle-EGFP-CMV, and the recombinant adenovirus plasmid pAdxsi-EGFP-CMV-hRAMP 1 was digested with XhoI to obtain 7 fragments, while the empty adenovirus plasmid as control only got 6 fragments. The recombinant adenovirus produced by the recombinant adenovirus plasmid packaged in 293 cells had pathogenicity to 293 cells, and the positive amplified bands of 164bp were identified by Ad-EGFP-hRAMP1 PCR of recombinant adenovirus. After repeated infection for many times, the titer of the recombinant adenovirus was 2.5 脳 10 11 PFU / ml. When the MOI value was 800, the transfection efficiency was 80%.RT-PCR to detect the mRNA expression of hRAMP1 gene in the transfected MSCs. Western blot was used to detect the bRAMP1 protein expression in the transfected MSCs. ConclusionThe recombinant adenovirus vector hRAMP1 carrying enhanced green fluorescent protein gene and hRAMP1 gene is an ideal gene vector cell and can be used for gene therapy of hRAMP1.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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