PSD-95及其突变体的重组腺病毒对皮质神经元的感染

目的构建PSD-95（postsynaptic density-95）及其突变体PSD-95Y523F和PSD-95Y533F（523和533位酪氨酸分别突变成苯丙氨酸）的复制缺陷型重组腺病毒载体，并检测腺病毒颗粒对体外原代培养皮质神经元的感染能力。方法双酶切将PSD-95及其突变体的基因分别从载体peDNA3．1（＋）亚克隆至穿梭载体pAdTrack—CMV中；电穿孔法将穿梭重组体转化到电转感受态细胞BJ5183中，使其与腺病毒骨架载体pAdEasy-1同源重组；同源重组体经PacI酶切线性化，脂质体法转入HEK293H细胞中进行病毒包装；病毒颗粒分别感染HEK293T细胞和原代皮质神经元，荧光检测或免疫印迹方法检测目的蛋白的表达水平。结果经酶切电泳鉴定和序列测定证明同源重组体中含序列正确的目的基因；重组腺病毒颗粒感染HEK293T细胞后，免疫印迹显示有较高水平的目的蛋白表达；重组腺病毒具有感染原代皮质神经元的能力，在未成熟神经元（体外培养5—9天）中感染率低，而在成熟神经元（体外培养13天）中感染率较高。结论成功构建了PSD-95、PSD-95Y523F、PSD-95Y533F的重组腺病毒载体；获得具有感染能力的重组腺病毒颗粒，易于感染成熟的神经元。

Adenovirus-mediated transfection with PSD-95 gene and its mutants in cultured cortical neurons

Objective To construct the replication -defective adenovirus recombinants encoding PSD -95, PSD - 95Y523F or PSD -95Y533F （tyrosine mutation to phenylalanine）, and to evaluate the transfection capacity of the recombinant virus particles in cultured primary cortical neurons. Methods The gene coding for PSD - 95, PSD - 95Y523F or PSD -95Y533F was subcloned from vector pcDNA3.1 （ ＋ ） into shuttle vector pAdTrack - CMV by double endonuclease restriction. The pAdTrack -CMV recombinants were introduced into E. coli BJ5183 ceils containing adenovirus backbone plasmid pAdEasy - 1 by electruporation to undergo homologous recombination. The identified pAdEasy recombinants were linearized by Pac I and then transfected into HEK293H cells with Lipofectamine 2000 for the package of adenovirus particles. HEK293T cells or cortical neuron cultures were infected with the recombinant adenovirus particles and the expression of target proteins were determined by fluorescence microscopy and/or immunoblot. Results The pAdEasy recombinants were confirmed by restriction endonuclease analysis and DNA sequencing. The target proteins were highly expressed in HEK293 cells. The high transfection efficiency was observed in mature cortical neurons instead of unmature neurons. Conclusion Recombinant adenoviruses vectors of PSD -95, PSD -95Y523F or PSD -95Y533F genes were successfully constructed and recombinant adenoviruse particles having infective capacity were obtained, which enables the infection of mature neurons.