分泌表达Aβ阻断肽的重组腺相关病毒的构建

Construction of recombinant adeno-associated virus system secreting expression of ABAD-DP

目的构建可稳定分泌表达ABAD-DP的重组腺相关病毒系统,并在细胞水平研究它与Aβ42的结合能力。方法应用分子克隆技术构建含有融合基因NT4-TAT-6His-ABAD-DP(NTA)的腺相关病毒载体。应用磷酸钙共沉淀法进行病毒包装。应用免疫细胞化学方法检测重组病毒在Hela细胞中的表达。应用免疫荧光技术检测重组病毒与Aβ42在Hela细胞中的结合作用。结果 pGEM-T Easy/ABAD-DP经EcoR I酶切后,可以得到96bp的目的片段,ABAD-DP基因经DNA测序证实与GenBank序列一致;重组质粒pSSGH/NTA经EcoRⅠ与BamHⅠ联合酶切,可以得到384bp的目的片段;成功获得滴度为3.01×109PFU/ml的重组病毒;含有6×His标签的重组病毒在Hela细胞中实现表达;通过免疫荧光技术,可以分别观察到重组病毒组、Aβ42组和重组病毒+Aβ42组的绿色、红色和黄色荧光。结论成功获得能够稳定表达ABAD-DP并能够在细胞内与Aβ42结合的重组腺相关病毒,为阿尔茨海默病的治疗提供新的思路。

Objective To construct the adeno-associated viral vector secreting and expressing ABAD decoy peptide（ABAD-DP）,and to observe its binding capacity to Aβ42 in Hela cells.Methods The fusion gene,NT4-TAT-6His-ABAD-DP（NTA）,was constructed using molecular biology methods.Hela cells were transfected with NTA,Aβ42 or both of them by recombinant virus.The expression of NTA,Aβ42 or both of them were detected by immunofluorescence.Results The fusion gene was identified by the restriction enzymes and DNA sequencing.NTA was stained green and Aβ42 was stained red in the cytoplasm of Hela cells.In the coinfection cells,NTA（green） and Aβ42 peptide（red） were identified co-localized（yellow）.Conclusions The rAAV/NT4-TAT-6His-ABAD-DP was confirmed stable expression of ABAD-DP,and NTA could bind Aβ42 peptide effective in cells.It may provide a promising candidate for the treatment of AD.