针对 MAG,OMgp,NogoA 基因的 scFv (OMgp)-9R-siRNA 复合物的制备和鉴定

摘要

目的 运用抗体靶向技术联合siRNA形成双特异性复合物scFv-9R-siRNA的高效递送系统,该系统通过抗OMgp的scFv把MAG,OMgp和Nogo A的特异性siRNA特异性运输至受损神经细胞,从而抑制这三种轴突抑制物的表达,改善脊髓损伤后的局部微环境,阻断这些髓磷脂相关抑制物对轴突再生的抑制作用,为后续的大鼠模型试验奠定基础.方法 (1)根据基因重组技术,利用制备好的高效抗OMgp单克隆抗体构建单链抗体scFv(OMgp),并在此基础上非共价合成针对MAG,OMgp,NogoA基因的scFv (OMgp)-9R-siRNA复合物;(2)体外鉴定scFv (OMgp)-9R-siRNA复合物效果:鉴定MAG,OMgp,NogoA三种抑制物的mRNA和蛋白表达水平,同时与非神经细胞对比,研究引入scFv后所产生的靶细胞特异性.结果 (1)成功制备抗大鼠OMgp单克隆抗体E811,并通过ELISA和Western blot对已制备的抗大鼠OMgp单克隆抗体E811进行了特异性和敏感性鉴定,在此基础上成功获得scFv (OMgp)-9R-siRNA复合物;(2)体外实验证实:scFv (OMgp)-9R-siRNA复合物同时特异性下调MAG,OMgp和Nogo A基因表达.结论 单链抗体技术(scFv)解决了siRNA的特异性导入受损神经元问题,为体内应用siRNA技术治疗脊髓损伤奠定了基础.%Objective The antibody targeted technical combined with siRNA was used to produce a bispecific complex scFv-9R-siRNA, which is a highly efficient delivery system. The delivery system would specifically transport the specific siRNAs of MAG, OMgp and NogoA to the injuried nerve cells through anti-Omgp scFv to inhibit the expression of these three axon-inhibitors, improve the local micro-environment after spinal cord injury, block the inhibition of these myelin-related inhibitors on axonal regeneration, and establish solid foundation for the subsequent rat model experiment. Methods (1) Single-chain antibody scFv (OMgp) was constructed with monoclonal anti-OMgp by recombinant DNA technology, and then, scFv (OMgp)-9R-siRNA was non-covalent synthesized for the genes of MAG, OMgp, NogoA; (2) Effects identification of scFv (OMgp)-9R-siRNA complex in vitro: mRNA and protein expression levels of MAG, OMgp and NogoA were identified in nerve cells comparing with non-nerve cells at the same time, to discover the cell-targeting specificity of scFv. Results (1) Rat OMgp monoclonal antibody E811 was successfully prepared, then specificity and sensitivity of that were idendified with ELISA and Western blotting. Finally we successfully got the scFv (OMgp)-9R-siRNA complex; (2) In vitro experiments confirmed that: scFv (OMgp)-9R-siRNA complexes simultaneously decreased the expression of MAG, OMgp and Nogo A specifically. Conclusion Single chain antibody technology resolved the specific import problems of siRNAs, and provided the foundation for the further study which is transporting the specific siRNA to damaged neurons in vivo and made axonal regeneration on the condition of spinal cord injury.