一种重组腺病毒载体产生及操作的新方法

目的建立一种快速、高效的产生腺病毒的实验方法。方法将增强型绿色荧光蛋白(EGFP)装在穿梭质粒，线性化后与腺病毒骨架载体一起电穿孔转染大肠杆菌，使之同源重组，产生病毒基因组质粒。后Pac酶切，脂质体介导转入293细胞以包装出病毒。扩增后收获病毒，氯化铯密度梯度离心纯化，空斑试验测滴度。结果52个大肠杆菌克隆，其中有1个发生同源重组，产生腺病毒基因组质粒pAdEGFP，转染293细胞，荧光显微镜下产生绿色荧光。病毒纯化后达10^11pfu／ml。结论大肠杆菌内质粒间同源重组的方法可以高效、简便、快捷地产生重组腺病毒载体，是一种很好的制备重组腺病毒载体的方法。

A Method for Production and Manipulation of Recombinant Adenovirus Vector

Objective To develop an experimental rapid and effective method for production of recombinant adenovirus vector. Methods The enhanced green fluorescent protein (EGFP) gene-exogenous gene was subcloned into the shuttle plasmid of the AdEasy adenovirus system. Then the resultant plasmid was lineated and subsequently cotransfected into the Escherichia Coli by electroporation together with the backbone plasmid pAdEasy 1. After their homologous recombination, the cloned recombinant adenovirus genome plasmid DNA was obtained, digested with Pac I to release ITR and transfected into package 293 cells with cationic liposome to get recombinant virus. Genomic homogeneous recombinant adenovirus was tested under electron microscopy for morphology and fluorescent microscopy was performed for detection of the expression of EGFP. Then the recombinant virus was propagated. After acquisition, it was purified by chlorinated cesium (CsCl) density gradient ultracentrifugation. Its titer was determined by plaque assays. Results After electroporation, there was only one clone that was proved to be successfully recombinated in the screened 52 Escherichia Coli clones. The cloned adenovirus genome plasmid pAdEGFP was obtained. After the plasmid DNA was transfected into 293 cells, the production of the recombinant adenovirus vector was observed under electron microscopy. They also expressed tremendous EGFP in 293 cells under fluorescent microscopy. The titer of virus stocks was up to 10 11 plaque forming units (pfu)/ml after purification. Conclusion Construction of recombinant adenovirus genome by homologous recombinant in Escherichia Coli is a very efficient, convenient and quick method to construct recombinant adenovirus.