分泌睫状神经营养因子腺病毒载体的构建及小量复制病毒增强其分泌表达的研究

Construction of Adenovirus Vector of Secretory Ciliary Neurotrophic Factor and Increasing of Its Secretion by Replicative Adenovirus

目的:探讨复制型腺病毒能否增强增殖缺陷型腺病毒Ad5-hCNTF所携带外源基因的表达分泌。方法:亚克隆获得分泌型睫状神经营养因子的基因(ciliary neurotrophic factor),然后将此基因插入到穿梭质粒pshuttle。pshuttle-hCNTF经pme1酶切后,CIAP去磷酸化,利用Ad-EASY腺病毒制备系统,将其与腺病毒骨架质粒pAdEasy-1共同转化大肠杆菌BJ5183,通过同源重组,筛选出含目的基因的重组型腺病毒质粒的菌株,获得大量该质粒后转染病毒包装细胞AD-293,成功包装出一种血清5型增殖缺陷型腺病毒Ad5-hCNTF。结果:经PCR鉴定该病毒含有该基因片断,Western blotting证实该病毒感染细胞后能表达CNTF蛋白。采用ELISA法检测培养液证实感染细胞能高水平地分泌CNTF。结论:体外实验表明在不同滴度的复制型腺病毒Ad5-E1+E3+的带动下,该病毒感染细胞后分泌表达目的蛋白的水平显著提高,为今后应用Ad作为基因治疗的载体提供实验证据。

Objective：We investigate whether replication-competent adenovirus Ad5（E1＋E3＋） could enhance the expression of hCNTF secreted by celllines after replication-defective adenovirus Ad5-hCNTF infection in vitro.Methods：Plasmid encoding human CNTF was digested and subcloned into shuttle plasmid pshuttle to obtain a recombinant plasmid pshuttle-hCNTF.Using Ad-EASY system,the lineared and dephosphorylated shuttle vector and pAdEasy-1 plasmid which contain most of the human adenovirus serotype 5 genome were cotransfomed into BJ5183 bacteria,a recombination event then take place and result in the production of recombinant AdEasy plasmid,then the plasmid carrying adenovirus major genome and target gene was transfected into package cells AD-293 celllines to obtain a replication-defective adenovirus Ad5-hCNTF.Results：The recombinant adenovirus Ad5-hCNTF were analyzed by PCR and the expression of hCNTF in retinal pigment epithelium（RPE） cells after Ad infection were examined by Western blotting,and the ability of the cell to secrete CNTF was measured with a sandwich enzyme-linked immunosorbent assay（ELISA）.the results indicated that Ad5-hCNTF contained the target gene,and were capable of infecting RPE celllines and produce the protein into the supernatant.Conclusion：We also have demonstrated that combined with different amount of replication-competent adenovirus Ad5（E1＋E3＋） in vitro,the expression of hCNTF secreted by celllines after Ad infection was enhanced significantly,and the enhancement of gene expression was observed in a concentration-dependent manner,and it offers basis for further study of gene therapy by Ad.