携带sFlt-1基因片段的慢病毒载体的构建及表达

Construction of lentiviral expression plasmid carrying different fragments of sFlt-1 gene

目的分别建立携带可溶性血管内皮生长因子受体-1(sFlt-1)不同基因片段的慢病毒表达质粒,为进一步研究sFlt-1不同基因片段表达的蛋白生物学功能奠定基础。方法以人新鲜胎盘组织提取的cDNA为模板,进行RT-PCR扩增,制备sFlt-1基因片段sFlt-1(2-3)和sFlt-1(2-4)。扩增片断经限制性内切酶BamHI和SalI双酶切后,插入慢病毒载体pRRL-GFP中,构建慢病毒表达质粒pRRL/sFlt-1(2-3)及pRRL/sFlt-1(2-4),与慢病毒包装载体pMDLg/pRRE、包被载体PRSV/REV、pMD2.G共转染人胚肾细胞(HEK293T),获得重组慢病毒Lenti.sFlt-1(2-3)及Lenti.sFlt-1(2-4),并感染人视网膜色素上皮(RPE)细胞,通过RT-PCR和Western blotting验证Lenti.sFlt-1(2-3)及Lenti.sFlt-1(2-4)在人RPE细胞的表达。结果经酶切鉴定及基因测序证实pRRL/sFlt-1(2-3)和pRRL/sFlt-1(2-4)的序列与GenBank中的序列完全一致,并且包装成病毒后能有效感染人RPE细胞。结论本实验成功构建了分别携带sFlt-l基因第2-3和第2-4免疫球蛋白样区域编码序列的慢病毒载体,包装成病毒后能有效感染人RPE细胞。

Objective sFh-1,a soluble secreted variant of the vascular endothelial growth factor （VEGF）receptor-1 that possesses only its extraeellular domain,is a specific endogenous inhibitor of VEGF. It is involved in retinal vasculogenesis. This study was to construct the lentiviral expression plasmids carrying different fragments of soluble fins-like tyrosine kinase-1 （ sFh-1 ） gene. Methods The target fragments, sFlt-1 （2-3） and sFlt-1 （2-4） ,were amplified by RT-PCR from fresh human placenta. Then the fragments were subcloned into the lentiviral vector,pRRL-GFP to generate the lentiviral expression vector,pRRL/sFlt-1 （2-3） and pRRL/sFlt-1 （2-4）. The corrected sFlt-1 gene fragments were confirmed by endoenzym digestion and sequencing. Recombinant lentiviruses were produced by HEK293T ceils following the co-transfection of pRRL/sFh-1 with the packaging plaslnids pMDLg/pRRE,PRSV/REV and pMD2. G. The resulting recombinant lentiviruses Lenti. sFlt-1 （2-3） and Lenti. sFlt-1 （2-4） were then used to infect human retinal pigment epithelial cells strain （D407）. SFh-I expression in retinal pigment epithelial cells was detected by RT-PCR and Western blotting analysis. Results The target gene fragments, sFlt-1 gene fragments including sFlt-1 （ 2-3 ） and sFlt-1 （ 2-4 ） , were successfully cloned from fresh human placenta at 633 bp and 915 bp with the same values and sequence to their theory fragments. Restriction enzyme digestion and nucleotide sequence analysis verified that the corrected fragments were ligated into pRRL-GFP lentiviral vector. The recombinant lentiviruses carrying sFlt-1 （2-3） or sFh-1 （2-4） could infect human retinal pigment epithelial cells, and expression of sFlt-1 was detected in retinal pigment epithelial ceils. Conclusion The recombinant lentiviruses can deliver target sFh-1 gene fragments to human retinal pigment epithelial cells and have high infection efficiency.