用大肠杆菌同源重组获得克隆化重组腺病毒基因组

利用大肠杆菌细胞内质粒间同源重组获得克隆化重组腺病毒基因组 DNA,高效构建携带有外源基因的均一重组腺病毒 .将带有狂犬病毒糖蛋白 (GP)基因和加强型 GFP(enhanced GFP,EGFP)表达盒的重组穿梭质粒 p Ad- Track- CMV/ GP与腺病毒骨架载体质粒 p Ad Easy- 1一起同时电击共转化大肠杆菌 BJ51 83.在 BJ51 83细胞内 ,带有同源序列的重组穿梭质粒与骨架载体可进行同源重组 ,得到以质粒形式存在的克隆化重组腺病毒基因组 p Ad- GP’.以 p Ad- GP’为模板 ,经DNA测序确认 GP基因成功整合入此质粒中的腺病毒基因组 E1区外源基因表达盒中 .线形化的p Ad- GP’转染 2 93细胞后可得到基因组结构均一、在 E1区插入有 GP和 EGFP表达盒的重组腺病毒 ,病毒滴度可达 1× 1 0 8pfu/ ml.电镜下此重组病毒颗粒直径约为 70 nm,略呈球形 ,用荧光显微镜观察感染细胞有很强的 EGFP表达 .实验表明 :利用大肠杆菌同源重组获得克隆化的重组腺病毒基因组 DNA,可高效制备高滴度的均一重组腺病毒

Construction of Cloned Recombinant Adenovirus Genome by Homologous Recombination in Escherichia coli

To efficiently construct cloned human type 5 recombinant adenovirus bearing exogenous rabies glycoprotein gene by employing homologous recombination in Escherichia coli .The rabies glycoprotein gene(GP)was first subcloned into shuttle vector pAd Track CMV in which an enhanced green fluorescent protein gene(EGFP)expression cassette had been incorporated in advance.The resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 cells by electroporation with adenoviral backbone plasmid pAdEasy 1.To directly confirm recombination,conventional DNA sequencing was performed with the double strand plasmid DNA as template and oligonucleotide synthesized as GP specific primer.After the cloned recombinant adenovirus plasmid DNA was obtained,it was digested with Pac I for releasing ITR and transfected into package 293 cell to get recombinant virus.Genomic homogeneous recombinant adenovirus was tested by electron microscope for morphology and fluorescent microscope for detection of the expressed EGFP.As a result,DNA sequencing indicated that GP gene was successfully integrated into the target region of the adenoviral genome.Homogenous recombinant virus could be easily obtained by transfecting 293 cell with this infectious DNA.The resultant recombinant adenonovirus could reach 1×10 8 pfu/ml in 293 cells and showed typical adenovirus morphological characteristics with a diameter about 70 nm when observed by electron microscope.They also expressed tremendous EGFP in infected cells.These experiments suggest that construction of cloned recombinant adenovirus genome by homologous recombination in Escherichia coli is a very efficient method to construct homogeneous recombinant adenovirus.