绿色荧光蛋白为报导基因快速筛选重组羊痘病毒表达载体

Rapid selection of recombinant orf virus expression vectors using green fluorescent protein

目的利用绿色荧光蛋白(GFP)为报导基因,构建通用、高度减毒的外源基因表达载体IA82△121-V。方法抽提病毒DNA,扩增ORFV132左右同源臂,构建穿梭质粒p SPV-132LF-EGFP-132RF;转染OFTu细胞,与羊痘病毒IA82△121同源重组,通过荧光信号筛选重组体IA82△121-V,PCR及测序鉴定,并经病毒滴定、体外血管内皮细胞增殖实验以检测ORFV132缺失后的功能变化。结果成功快速筛选出重组羊痘病毒IA82△121-V,初步判断ORFV132的缺失不影响病毒体外的生长复制功能,但是减轻了其毒力。结论利用G FP为报导基因能简单、快速、稳定地筛选出重组羊痘病毒,高度减毒的重组羊痘病毒IA82△121-V有望成为新一代外源基因表达载体。

Objective To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein（GFP） as the reporter gene. Methods The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid p SPV- 132LF- EGFP- 132 RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82△121 by homologous recombination. The recombinant IA82△121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. Results The recombinant orf virus IA82 △ 121- V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. Conclusion Green fluorescent protein is a selectable marker for rapid,convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82△121-V can serve as a new expression vector for exogenous genes.