人LMP-1基因的腺病毒重组体构建及在BMSCs的表达

发布时间：2018-04-01 04:38

本文选题：LIM矿化蛋白　切入点：腺病毒　出处：《重庆医科大学》2007年硕士论文

【摘要】： 目的:应用AdEasy腺病毒载体系统,构建人LMP-1基因的腺病毒重组体,感染体外培养的兔BMSCs,鉴定细胞对LMP-1的表达情况,为进一步动物实验奠定基础。方法:采用PCR的方法以pIRES2-EGFP-LMP-1质粒为模板进行扩增,在下游引物中加入特定序列,使扩增出的LMP-1基因带有编码6个组氨酸(即His标签)的碱基序列。将得到的带有His标签碱基序列的LMP-1基因作为目的基因,通过TA克隆与pMD18-T载体连接并测序。双酶切后插入至腺病毒穿梭质粒pAdtrack-CMV内。PmeⅠ线性化阳性克隆pAdtrack-LMP-1,在BJ5183菌内完成与骨架质粒pAdeasy-1的同源重组,构建出重组腺病毒质粒pAd-LMP-1。通过脂质体介导在HEK293细胞内包装出复制缺陷的重组腺病毒Ad-LMP-1,大量扩增、纯化并测定滴度。结合骨髓密度梯度离心法和贴壁筛选法从兔骨髓中分离培养BMSCs。重组腺病毒以不同的MOI感染第三代BMSCs,在显微镜下观察其生长情况及感染效率。然后以最佳MOI感染BMSCs,3天后收集细胞行RT-PCR法检测LMP-1基因的mRNA表达,再利用抗His标签的特异抗体以Western Blot的方法鉴定LMP-1基因的蛋白表达。结果:测序鉴定携带His标签的LMP-1基因的重组质粒pMD18-T构建成功。成熟的体外分离培养BMSCs的方法方便简单,收获细胞数量多且增殖能力强。重组腺病毒以150的MOI值感染兔BMSCs可以获得最佳的感染效率,即在3天后达到50%～70%。收集病毒感染后的BMSCs行RT-PCR检测表明存在LMP-1基因的mRNA表达,Western Blot亦证实LMP-1基因的蛋白表达。结论:成功构建携带His标签的人LIM矿化蛋白-1(LMP-1)基因的腺病毒重组体。证实重组病毒感染BMSCs后细胞能有效地表达LMP-1,为LMP-1动物体内的实验研究提供了依据。
[Abstract]:Objective: to construct adenovirus recombinant of human LMP-1 gene by AdEasy adenovirus vector system, and infect rabbit BMSCs in vitro, identify the expression of LMP-1, and lay a foundation for further animal experiments.
Methods: the PCR method using pIRES2-EGFP-LMP-1 plasmid as template for amplification, adding specific sequence in the downstream primers, the LMP-1 gene was amplified with encoding 6 histidine (His tag) of the base sequence. The His tagged nucleotide sequence of the LMP-1 gene as the target gene, was cloned by TA and pMD18-T vector. Double enzyme digestion and sequencing. After inserted into Adenovirus Shuttle Plasmid pAdtrack-CMV.Pme 1 linear positive clone pAdtrack-LMP-1 BJ5183 in bacteria within homologous recombination with plasmid pAdeasy-1, the recombinant adenovirus plasmid pAd-LMP-1. by liposome mediated package of recombinant adenovirus Ad-LMP-1 replication defective in HEK293 cells proliferation purification, and the titer was determined.
And adherence screening method from rabbit bone marrow in the separation of the third generation BMSCs were infected with different MOI BMSCs. recombinant adenovirus combined with cultured bone marrow by density gradient centrifugation, to observe the growth and infection efficiency under the microscope. Then the optimal MOI BMSCs infection, collected 3 days after the expression of RT-PCR was detected by LMP-1 gene of mRNA cell line, and then the use of specific antibody anti His tag with LMP-1 Western Blot method for identification of gene expression.
Results: the recombinant plasmid pMD18-T LMP-1 gene sequencing with His tag were successfully constructed. The mature in vitro culture method of BMSCs is convenient and simple, the cells were harvested and a large number of strong proliferation ability. The recombinant adenovirus with MOI values of 150 rabbits infected with BMSCs can get the best effect of infection rate reached 50% ~ 70%. BMSCs collection virus RT-PCR detection showed that the expression of LMP-1 gene mRNA in 3 days, Western Blot also confirmed the expression of LMP-1 protein.
Conclusion: the adenovirus recombinant carrying human His LIM protein -1 (LMP-1) gene was successfully constructed. It is proved that the recombinant LMP-1 can effectively reach LMP-1 after infection with BMSCs, which provides a basis for the experimental research of LMP-1 animal.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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