利用电穿孔法对狂犬病病毒SRV9 Ψ区缺失株病毒的拯救及鉴定  

Rescue and Identification of Rabies Virus SRV9 Ψ Aera Delection strain by Electroporation Transfection

作　　者：毛丽萍[1] 魏玉圆 王伟[1] 翟少华[1] 程瑶[1] 文兆海 简子健[1] 

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《动物医学进展》2017年第11期22-27,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31360623);研究生科研创新项目(XJAUGRI2015017)

摘　　要：探讨电穿孔转染法对SRV9Ψ区缺失株重组病毒的拯救及鉴定。利用电穿孔细胞转染法,将纯化的Ψ区缺失株pcDNA-NPMG-L全长质粒和pcDNA-N、pcDNA-P、pcDNA-M、pcDNA-G和pcDNA-L辅助表达质粒共转染至BHK-21细胞中,并进行盲传,借助RT-PCR、间接免疫荧光试验、Western-blot及透射电子显微镜对重组病毒进行鉴定。结果显示,重组病毒能够产生绿色荧光,透射电子显微镜观察可见典型的弹状病毒,对重组毒株与亲本毒株G蛋白进行Western-blot验证,可获得相应目的条带。结果表明,通过电穿孔细胞转染法,能成功拯救出重组病毒SRV9Ψ区缺失株,为病毒的拯救提供新的转染方法,并能有效提高转染效率,为研发新型狂犬病疫苗提供参考。The aim of the study was to investigate the rescue and identification of recombinant virus of SRV9 without pseudo gene Ψ region via electroporation transfection.The purified deleted pseudo gene region pCDNA-NPMG-L and the helper plasmids pcDNA-N,pcDNA-P,pcDNA-M,pcDNA-G and pcDNA- L were co-transfected into BHK-21 cells via electroporation transfection for virus rescue,and the BHK21 cells were used for blind passages of the recombinant rabies virus.The recombinant rabies virus SRV9 deleted pseudo gene Ψ region was identified by RT-PCR,indirect immunofluorescence,Western-blot and electron microscopy.The results showed that the recombinant virus could produce green fluorescence in the indirect immunofluorescence assay,the typical rhabdovirus was observed by transmission electron microscopy.The recombinant strain and the parent strain G protein were subjected to Western-blot to obtain the corresponding target band.The above results showed that transfection through electroporation cells in this experiment was able to rescue its recombinant virus SRV9 Ψ region deletion strain.This study provided a new transfection method for rescue of virus, and can effectively improve the transfection efficiency, and provide effective theoretical basis and practical basis for the development of new rabies virus vaccine.

关 键 词：狂犬病病毒 电穿孔转染法 SRV9Ψ区缺失株 拯救 

分 类 号：S852.659.6[农业科学—基础兽医学]