人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒的构建及活性检测

背景：前期试验显示单个病毒载体介导的多基因共表达系统能够显著提高椎间盘退变转基因疗效。目的：构建人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒。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Lenti-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mRNA和蛋白质表达水平。结果与结论：RT-PCR和Western-blot技术检测结果显示重组质粒成功表达了3种目的基因,并于转染后48h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%。说明成功构建了携带人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因的慢病毒多表达质粒。

Construction and detection of lentiviral plasmids containing human transforming growth factor beta 3, connective tissue growth factor and tissue inhibitor of metalloproteinases 1 gene

BACKGROUND： The former tests showed that multi-gene co-expression system mediated by virus can significantly improve the transgenic curative effect of intervertebral disc degeneration. OBJECTIVE： To construct the recombinant plasmid of lentivirus containing human transforming growth factor beta 3 （TGFβ3）, connective tissue growth factor （CTGF） and tissue inhibitor of metalloproteinases 1 （TIMP1）. METHODS： 2A self-cleaving sequences were used to ligate the cDNA of TGFβ3, CTGF, TIMP1 in a single open reading frame derived from plasmid of lentivirus in order to obtain the recombinant lentiviral plasmid Lenti-TGFβ3-P2A-CTGF-T2A-TIMP1. RT-PCR and Western-blot were used to detect mRNA and protein expressions of TGFβ3, CTGF, TIMP1 in different times after transfection. RESULTS AND CONCLUSION： RT-PCR and Western-blot detection shows that the mRNA and protein expressions of TGFβ3, CTGF, TIMP1 were detected in transfected 293 cells, the protein expressions reached peak value at 48 hours after transfection, and the protein level of downstream gene was about 80% of upstream gene. The recombinant plasmid, Lenti-TGFβ3-P2A-CTGF-T2A-TIMP1, is constructed successfully, providing a basic model for the packaging of virus and further study on therapy of intervertebral disc degeneration.