人TGFb3、CTGF和TIMP1基因重组慢病毒多表达质粒的构建及活性检测

发布时间：2018-01-28 14:24

本文关键词： TGF β3 CTGF TIMP1 慢病毒 293细胞　出处：《青岛大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：构建人转化生长因子β3(TGFβ3)、结缔组织生长因子(CTGF)和基质金属蛋白酶抑制剂(TIMP1)基因重组慢病毒多表达质粒。转染293细胞后检测活性、包装病毒并探讨其作用特点。方法：应用全基因合成技术,以“自剪切多肽2A”串联目的基因,并克隆到慢病毒表达质粒构建重组慢病毒质粒pLVX-TGF β3-P2A-CTGF-T2A-TIMP1。转染293细胞后,应用RT-PCR和Western-Blot技术分别检测转染后不同时间点目的基因的mRNA和蛋白质表达水平。结果：RT-PCR和Western-blot技术检测结果显示重组质粒成功表达了目的基因并于转染后48小时左右达到峰值,“2A”结构下游基因蛋白质表达量约为上游基因的80%。经慢病毒包装后获得5X108TU/ml滴度的活性病毒。结论：成功构建了携带人TGFβ3、CTGF和TIMP1基因的慢病毒多表达质粒并收获较高滴度的活性病毒,为转染椎间盘髓核细胞和动物实验打下基础。
[Abstract]:Objective: to construct recombinant lentivirus multi expression plasmid of human transforming growth factor beta 3 (TGF beta 3), connective tissue growth factor (CTGF) and matrix metalloproteinase inhibitor (TIMP1). After transfection into 293 cells, we detected the activity, wrapped the virus and discussed its action characteristics.
Method: using the gene synthesis technology, with "self splicing polypeptide 2A" tandem gene, and cloned into the lentiviral expression plasmid to construct the recombinant lentiviral plasmid pLVX-TGF beta 3-P2A-CTGF-T2A-TIMP1. transfected 293 cells after transfection were detected after different time points of target gene and protein expression levels of mRNA using RT-PCR and Western-Blot technology.
Results: the RT-PCR and Western-blot results showed that the recombinant plasmid gene was expressed successfully in 48 hours after transfection and reached the peak, the "2A" structure of downstream gene protein expression about live viral upstream gene 80%. by lentiviral packaging after 5X108TU/ml titer.
Conclusion: the lentivirus multi expression plasmid carrying human TGF beta 3, CTGF and TIMP1 genes was successfully constructed, and the higher titer of the active virus was harvested, which laid the foundation for transfection of intervertebral disc nucleus pulposus cells and animal experiments.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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