负载紫红质通道蛋白2重组腺病毒的包装及功能鉴定

Package and identification of replication deficient recombinant adenovirus expression vector of channelrhodopsin-2

背景紫红质通道蛋白2（ChR2）是从单细胞绿藻莱茵衣藻上分离的一种光敏感通道蛋白，可作为光刺激神经细胞的手段，与电、磁和超声波相比，光在时间和空间上对神经细胞的刺激将更精确。目的构建负载ChR2基因的重组腺病毒，并鉴定其功能。方法将人胚肾293（HEK293）细胞在含质量分数10％胎牛血清的DFl2培养基中进行培养及传代。腺病毒穿梭质粒pSB291-hCHR2-GFP与腺病毒包装质粒pBHGloxAEI，3Cre共转染HEK293，包装得到少量负载ChR2基因的重组腺病毒（Ad—ChR2），经HEK293细胞扩增、CsCl梯度离心，Tris—HCl透析后得到纯化Ad—ChR2。获取4只出生1d的清洁级LongEvans大鼠视皮层组织，用组织块培养法利用无血清培养基原代培养视皮层细胞，用纯化的Ad—ChR2转染培养的视皮层细胞，当转染成功的视皮层细胞表达绿色荧光时给予460hill蓝光刺激，应用膜片钳技术记录视皮层细胞的动作电位。结果纯化浓缩后的Ad—ChR2滴度可达7．9×10^10PFU／ml，HEK293细胞转染Ad—ChR224h后倒置荧光显微镜下即可见细胞膜上有绿色荧光表达，转染13d可见细胞由扁平变为圆形。无血清培养的视皮层细胞转染纯化的Ad—ChR2后细胞膜上可见绿色荧光蛋白（GFP）的表达，460nm蓝光刺激后用膜片钳技术可记录到蓝光诱发的视皮质细胞的动作电位。结论本研究成功构建了负载ChR2基因的重组腺病毒表达载体，并证实ChR2能够感染有功能的视皮质细胞，这对视觉可塑性方面的研究非常重要。

Background Channelrhodopsin-2 （ChR2）is a cation channel isolated from the eyespot of Chlamydomonas algae and has been used to control neuron activity. The light stimulation is a more precise fashion whether space or time than that of electrical, magnetic and ultrasound stimulation. Objective This study was to construct a replication deficient recombinant adenovirus expression vector of ChR2 and to determine its function. Methods Human embryo kidney 293 （HEK293）cell line was cultured and passaged in DF12 medium containing 10% fetal bovine serum（FBS）. The ChR2 gene was cloned at the downstream of cytomcgalovirus（CMV） promoter of the adenoviral shuttle plasmid pSB291 in sense direction,and the resultant recombinant plasmid pSB291-hChR2- GFP was transfected into HEK293 cell together with plasmid pBHG lox （ deltaE1,3 ） containing adenoviral genome, then small amounts replication deficient recombinant adenovirus expression vector of ChR2 （ Ad-ChR2 ） was obtained. Through amplification gradient centrifugation and dialysis, pure Ad-ChR2 was obtained. Visual cortex cells derived from 4 1-day-old clean Long Evans rats were primary cultured with serum-free culture media and infected by Ad- ChR2. When expressing green fluorescence,those cells received the stimulated of blue light with 460 nm. Patch clamp technique was applied to record an action potential. Results After purification and concentration,the titer of Ad- hCHR2 reached 7.9 x 101~ PFU/ml. Twenty-four hours after transfect of Ad-ChR2, HEK293 cell membrane showed the green fluorescence for the recombinant plasmid with green fluorescence protein under the inversed fluorescence microscope. The HEK293 cells change their shape from flat to round 13 days after transfeeted. The primary cultured visual cortex cells exhibited the green fluorescence 3-5 days after infected by Ad-ChR2. The action potentials evoked by blue light stimulation were recorded with patch clamp on those cells expressing green fluorescence. Conclusions Ad-ChR2 expressing vector is constructed successfully in this study. It is verified that Ad-ChR2 expressing vector can infect visual cortex cells with visual function. This result is very important for visual plasticity study.