含人神经营养素-3的重组腺病毒的高效制备

Efficient Generation of Recombinant Adenoviruses Expressing Human Neurotrophin-3

目的采用改良的细菌内同源重组法快速高效构建含人神经营养素-3(neurotrophin-3,NT-3)的重组腺病毒,为其用于神经性聋的在体研究做准备。方法将人全长NT-3基因亚克隆至带绿色荧光蛋白(greenflu-orescent protein,GFP)的穿梭质粒pshuttle-IRES-hrGFP-1上,用电穿孔法使线性化的重组穿梭质粒与预转入腺病毒骨架pAdeasy-1的大肠杆菌BJ5183-AD-1同源重组,脂质体法转染AD293细胞并大量扩增、柱层析纯化得到重组腺病毒pAdeasy-NT-3,GFP测定病毒滴度。结果PCR检测证明人NT-3已整合入腺病毒基因组并表达;Western blot证实在感染重组腺病毒pAdeasy-NT-3的Hela细胞中有相应NT-3蛋白表达;病毒滴度分别为pAdeasy-NT-3109U/ml、pAdeasy-hrGFP1010U/ml。结论利用新型的腺病毒载体Adeasy XL系统可以快速构建同时表达GFP和NT-3的重组复制缺陷型腺病毒pAdeasy-NT-3,柱层析纯化法能制备高纯度的病毒。

Objective To construct recombinant adenovirus encoding human neurotrophin - 3（NT- 3） by using a high efficient method of homologous recombination in bacteria for treating sensorineural hearing loss in vivo. Methods Full length NT -3 cDNA was subcloned into a pshuttle- IRES- hrGFP- 1 vector. The recombinant plasmid （pshuttle- IRES- hrGFP- 1/NT - 3） was transformed into ultracompletent BJ5183 - AD - 1 cells containing bone plasmid pAdeasy - 1 to produce adenovirus vector encoding NT - 3 cDNA. Then the adenoviras vector was transferred into AD293 cells by liposome to get recombinant adenovirus particles . Adenovirus particles were purified by column chromatography and titered by GFP detection. Results PCR test indicated that the recombinant adenoviras contained the NT - 3 gene. Meanwhile, protein of NT - 3 was detected in the infected Hela cells by Westem blot. The titer of purified recombinant adenoviras： pAdeasy- NT- 3 10^9 U/ml, pAdeasy - hrGFP 10^10 U/ml. Conclusion Using the Adeasy XL system, the human NT - 3 recombinant adenoviras can be produced efficiently in AD293 cells. Adenovirus with high purity can be harvested by column chromatography.