应用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒

目的：AdEasy系统可在原核细胞E．coli BJ5183中完成穿梭质粒与骨架质粒之间的高效同源重组，经抗生素培养板筛选重组体，然后转染293细胞获得重组病毒，极大简化了载体的构建过程。实验利用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒。方法：实验于2006—08／2007—05在青岛大学医学院病毒学实验室完成。①实验材料：清洁级SD大鼠5只，实验过程中对动物的处置符合动物伦理学标准。腺病毒穿梭质粒pAdTrack—CMV，骨架质粒pAdeasy—1，大肠杆菌BJ5183和DH10B，人胚肾293细胞均由青岛大学医学院微生物教研室罗兵教授惠赠。②实验方法；从脂多糖刺激培养的大鼠脾细胞中提取细胞总RNA，应用反转录多聚酶链反应技术扩增白细胞介素10cDNA，定向亚克隆至pAdtrack—CMV中构建腺病毒穿梭质粒pAdtrack-CMV-IL-10，酶切线性化后转化到含腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌中，获得重组腺病毒质粒pAd-IL-10，Lipofectamin包装转染293细胞，获得重组腺病毒vAd—IL—10，荧光显微镜下观察绿色荧光蛋白的表达。将293细胞接种丁96孔板中，每孔1×10^4个细胞，浓缩后的病毒贮存液按不同比例稀释加至培养板中，测定重组腺病毒滴度。用重组腺病毒vAd—IL-10感染293细胞3d后，以TRIzol一步法提取细胞总RNA，所获RNA加入DNaseⅠ消化处理可能污染的DNA后，PCR产物行琼脂糖电泳检测白细胞介素10mRNA的表达。结果：①重组腺病毒的包装及滴度测定：经限制性内切酶转染293细胞16h后，荧光显微镜观察到细胞中有GFP荧光，可间接反映目的基因的表达。将病毒上清反复感染293细胞扩增重组腺病毒，通常传至第4～5代于接种病毒后24—48h，几乎可观察到所有细胞出现荧光，此时收获病毒，重组病毒命名为vAd—IL-10。vAd—IL—10滴度为5．5×10^8pfu／mL，vAd—GFP滴度为9．0×10^8pfu／mL?

Constructing recombinant adenovirus carrying rat interleukin-lO gene using AdEasy system

AIM： The homologous recombination takes place in E.coli BJ5183 between shuttle vector and adenoviral backbone vector. Recombinants are selected for kanamycin resistance, and the linearized recombinant plasmid is transfected into 293 cells. In this study, AdEasy system was used to generate recombinant adenovirus vector carrying rat interleukin-10 （IL-10） gene.
METHODS： The experiment was conducted in Department of Microbiology, Qingdao University Medical College from August 2006 to May 2007. ①The materials included five clean-grade SD rat, a shuttle vector pAdTrack-CMV, an adenoviral backbone plasmid pAdEasy-1, E. coli. BJ5183 and DHIOB, and human embryo kidney 293 cells, which were given as a present by professor Luo. All animal treatment was accorded with the ethical standards. ②Total RNA was extracted from spleen cells of rat stimulating by lipopolysaccharide. IL-10 cDNA was amplified by using RT-PCR. The gene of interest was firstly cloned into a shuttle vector pAdTrack-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease Pine Ⅰ, and subsequently transformed into E. coli. B J5183 cells with an adenoviral backbone plasmid pAdEasy-Ⅰ. Finally, the linearized recombinant plasmid was transfected into adenovirus packaging cell lines 293 cells. Recombinant adenovirus vAd-IL-10 was obtained. The expression of green fluorescent protein （GFP） was observed under fluorescent microscope. 293 cells were cultured in 96-well culture plate with 1×10^4 cells per well. Condensed virus stock solution was added into the plate at different ratios. Recombinant adenoviruses titer was determined. Three days later, total RNA was extracted from 293 cells by TRIzol one-step method, and contaminant DNA was digested by DNase Ⅰ . Electrophoresis detected the expression of IL- 10 mRNA.
RESULTS： ①GFP expression was observed 16 hours after packing of the linearized recombinant adenoviruses in 293 cells. The amplification product of replicationdeficient Ad-IL-10 virus was transfecte