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Construction of eukaryotic expression plasmid about human retina- derived NT-3 and its expression, secretion |
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| Construction of eukaryotic expression plasmid about human retina- derived NT-3 and its expression, secretion |
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作者：彭春霞 文章来源：首都医科大学 点击数：347 更新时间：2012/9/13 
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Purpose To construct recombinant plasmid containing gene of human neurotrophin-3(NT-3)-green fluorescent fusion protein (GFP) and to study the expression and secretion of NT-3-GFP fusion protein in cell and in media.
Method NT-3 genes were cloned by RT-PCR from fresh human retinal tissue and recombined into plasmid,PEGFP-N1 to construct PEGFP-N1-NT-3 plasmid with expressing NT-3-GFP fusion protein. Then, the PEGFP-N1-NT-3 vectors were transfected into 293T cells,following counting transfection efficiencies by fluorescent microscopy and
Image-Pro Plus6.0 Microsoft. The expression and secretion level of NT-3- GFP fusion protein in 293T cells and media was assayed respectively by Western-blot and ELISA.
Result 24 hours and 48 hours after transfection, the expression of NT-3-GFP was observed in cytoplasm and nucleus by fluorescent microscopy and laser scanning confocal microscopy.At 48 hours after transfection, the transfection efficiency was 50.01%. Results of Western-blot showed that pro-NT-3-GFP fusion protein of 54.5KDa size (proNT-3:27.5KDa+GFP: 27 KDa =54.5KDa) was detected and results of ELISA indicated that the concentration of mature NT-3-GFP fusion protein in media was 22.18ng/ml.
Conclusion The PEGFP-N1-NT-3 plasmid constructed can express and secret human NT-3-GFP fusion protein high level and it would provide potent technique supports for studying the neuroprotection of NT-3 for retinal cells and central neuron cells. Meanwhile it could make good preparation for assembling NT-3 ECT-based intraocular implants for treatment of retinitis pigmentosa(RP). |
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