| 摘 要: 目的：通过构建耳蜗毛细胞重组Prestin腺相关病毒-2(AAV-2)载体,为耳蜗Prestin蛋白调控研究提供工具。方法：以豚鼠Prestin基因为模板,PCR扩增与提取,利用基因工程技术构建AAV-2-Prestin载体并转染培养的耳蜗毛细胞,再采用Western blot检测转染后第1d、3d、5d耳蜗毛细胞Prestin蛋白的表达水平。结果：构建pAAV-EGFP-Prestin原病毒质粒转染HEK-293T细胞后,第1d可见微弱绿色荧光蛋白表达,第3d可见绿色荧光蛋白表达达到高峰；AAV-2-Prestin病毒转染染HEI-OC1细胞后,不同转染时间的各组Prestin蛋白表达水平有显著性差异(F=414.755,P＜0.001),3d和5d组Prestin蛋白表达显著高于1d(P＜0.001),3d组Prestin蛋白表达显著高于5d组(P＜0.001)。结论：重组腺相关病毒-2-Prestin载体成功构建，具有较好的转染效率和持续性。 |
| 关键词: 外毛细胞 腺相关病毒 Prestin |
|
中图分类号:
文献标识码:
|
| 基金项目: |
|
|
|
Lin Shou-kai, Wang Jun-yi, Wu Zhi-dan, Wu Shan
|
|
School of Public Health,Guangdong Pharmaceutical University
|
| Abstract: OBJECTIVE: To provides a tool for the study of cochlear Prestin protein regulation, the cochlear hair cell recombinant Prestin adeno-associated virus-2 (AAV-2) vector was constructing. METHODS: The guinea pig Prestin gene was used as a template for PCR amplification and extraction. The AAV-2-Prestin vector was constructed by gene engineering technology and transfected into cultured cochlear hair cells,and the expression level of Prestin was detected by Western blot on the 1st, 3rd and 5th day after transfection.RESULTS: After transfecting HEK-293T cells with pAAV-EGFP-Prestin proviral plasmid, the expression of weak green fluorescence was observed on the 1st day, and the expression of green fluorescence was peaked on the 3rd day. After transfection of HEI-OC1 cells with AAV-2-Prestin virus, the expression levels of Prestin protein were significantly different in different transfection time groups (F=414.755, P<0.001), and the expression of Prestin protein in 3d and 5d groups was significantly higher than 1d ( P<0.001), the expression of Prestin protein in the 3d group was significantly higher than that in the 5d group (P<0.001). Conclusion: The recombinant adeno-associated virus-2-Prestin vector was successfully constructed and it has good transfection efficiency and persistence. |
| Keywords: Outer hair cells Adeno-associated virus Prestin |
