基于λ-Red重组酶系统的Ⅳa2基因缺失及重组腺病毒的包装

Deletion of Ⅳa2 Gene from Adenoviral Genome by λ-Red Recombinase System and Packaging of the Recombinant Adenovirus

本研究用λ-Red重组酶介导的PCR打靶方法缺失腺病毒基因组上Ⅳa2基因的大部分编码序列(1104 bp),并获得一种Ⅳa2基因缺失的重组腺病毒。首先构建PCR打靶的含有卡那霉素抗性表达盒的模板质粒pAK,再以pAK为模板扩增出两端含39 bp同源臂的线性DNA片段;将pFG140质粒及线性DNA片段依次转化到宿主菌BW25113/pIJ790中,通过λ-Red重组酶介导的同源重组获得了缺失Ⅳa2基因的腺病毒基因组质粒pFG140-ΔⅣa2(1104)。酶切及DNA测序结果显示,用λ-Red重组酶介导的PCR打靶方法在pFG140上精确地缺失了预计的Ⅳa2基因3'端的1104bp片段。用PCR方法扩增获得Ⅳa2基因全长ORF,插入表达载体pAAV2neo中,构建成Ⅳa2基因表达质粒pAAV2neo-Ⅳa2。将pFG140-ΔⅣa2(1104)与pAAV2neo-Ⅳa2共转染HEK293细胞,成功地获得了重组腺病毒Ad5ΔⅣa2(1104)。Western Blot的结果表明,Ad5ΔⅣa2(1104)病毒感染的HEK293细胞检测不到Ⅳa2蛋白的表达。本研究建立了一种对腺病毒基因组直接进行缺失操作的λ-Red重组酶介导的PCR打靶方法,并获得了一种IVa2基因大部分缺失的重组腺病毒。

This investigation is to delete the most of the coding sequence（1 104 bp） of the Ⅳa2 gene in an adenovirus genome by a λ-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with Ⅳa2 deletion.First,the template pAK of PCR targeting,containing kanamycin cassette,was constructed.Then,a linear fragment for PCR targeting,which had 39 bp homologous arms at both of its terminus,was amplified by PCR from the pAK.The pFG140 and the linear fragment were electroporated into E.coli BW25113/pIJ790 sequentially and the recombinant pFG140-ΔⅣa2（1104） was established by homologous recombination between the linear fragment and the pFG140 with aid of λ-Red recombinase.The precise deletion of 1 104 bp fragment from Ⅳa2 was confirmed by restriction endonucleases digestion and DNA sequencing.ORF of Ⅳa2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector.The recombinant adenovirus Ad5ΔⅣa2（1104） was rescued by co-transfection of pFG140-ΔⅣa2（1104） and pAAV2neo-Ⅳa2 into HEK293 cells.It was shown by Western Blot that Ⅳa2 could not be detected in the Ad5ΔⅣa2（1104）-infected HEK293 cells.This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a λ-Red recombinase system,and a recombinant adenovirus with Ⅳa2 deletion was obtained.