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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: To investigate whether replication-competent adenovirus can enhance the expression and secretion of exogenous genes of adenovirus Ad5-hCNTF. Methods: The gene of secreting ciliary neurotrophic factor was subcloned and then inserted into the shuttle plasmid pshuttle. After digestion of pshuttle-hCNTF with pme1, CIAP was dephosphorylated and transformed into E.coli BJ5183 by adenovirus backbone plasmid pAdEasy-1 by using Ad-EASY adenovirus preparation system. Through homologous recombination, the target gene was screened Recombinant adenovirus plasmid strain, obtained a large number of the plasmid transfected virus packaging cells AD-293, successfully packaged a serotype 5 proliferation-deficient adenovirus Ad5-hCNTF. Results: The virus was identified by PCR. The result of Western blotting showed that the virus could express CNTF protein after it was infected. The culture medium of ELISA confirmed that the infected cells could secrete CNTF at a high level. CONCLUSION: In vitro experiments showed that the target protein secreted by Ad5-E1 + E3 + cells infected with different titer of Ad5-E1 + E3 + could significantly enhance the expression of target protein and provide experimental evidence for the future application of Ad as gene therapy vector.