柔性肽串联四价Aβ_(15)重组腺病毒的构建及纯化

Construction and purification of flexible peptide linked quadrivalent Aβ_(15) adenovirus

目的构建并纯化含有柔性肽串联四价β-淀粉样蛋白(Aβ15)的重组腺病毒疫苗,用于老年性痴呆的预防和治疗。方法酶切获取带有IgGк轻链信号肽序列的柔性肽串联四价Aβ15基因,定向克隆于穿梭质粒pAdTrack-CMV中。通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-4×Aβ15,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-4×Aβ15)。Western检测4×Aβ15在培养液中的分泌表达。氯化铯密度梯度离心纯化重组腺病毒Ad-4×Aβ15。结果 pAdTrack-4×Aβ15测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-4×Aβ15,western检测4×Aβ15在培养上清中表达。经氯化铯密度梯度离心纯化后Ad-4×Aβ15病毒颗粒数约为4.6×1012 VP/mL。结论成功制备了重组腺病毒Ad-4×Aβ15,感染细胞证实其呈分泌表达,经氯化铯密度梯度离心纯化后得到高滴度的重组腺病毒。为老年性痴呆(Alzheimer’s disease,AD)的实验治疗打下基础。

Objective To construct and purify the flexible peptide linked quadrivalent Aβ15 Adenovirus vaccine for preventing and therapy of the Alzheimer disease（AD）.Methods The IgG к light chain fused flexible peptide linked quadrivalent Aβ15 sequence was acquired by restricted enzyme digestion.And it was cloned to the shuttle plasmid pAdTrack-CMV.The homologous recombination between the recombinant vector and the adenovirus bone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector pAd-4×Aβ15.The infecting recombinant virus particles Ad-4×Aβ15 was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.Western blotting detected the secreted expression of 4×Aβ15 protein in the supernatant.Ad-4×Aβ15 was purified by CsCl2 density gradient ultracentrifuge.Results The sequence results of pAdTrack-4×Aβ15 were consistent with expectancy.After homologous recombination and packaging with 293 cells,the recombinant Ad-4×Aβ15 adenovirus was obtained.Western blotting suggested that the 4×Aβ15 protein was expressed secretly in the supernatant.After CsCl2 density gradient ultracentrifuge,the particles of Ad-4×Aβ15 adenovirus were about 4.6×1012 VP/mL.Conclusions The Ad-4×Aβ15 was constructed successfully.It is confirmed that the secreted interest proteins were expressed in the infected cells.After CsCl2 density gradient ultracentrifuge,the high titre Ad-4×Aβ15 adenovirus were obtained,which is important for AD experimental therapy.