摘要
为了利用CRISPR/Cas9技术高通量筛选介导猪瘟病毒入侵的功能性受体,试验首先用融合PCR技术,将Venus基因融合至Npro蛋白的第13位与14位氨基酸之间,获得全长感染性克隆pCSFVVenus;之后将pCSFV-Venus转染至SK6细胞拯救重组猪瘟病毒(rCSFV-Venus),并将拯救的病毒感染猪肾细胞(PK-15细胞)进行连续传代观察其稳定性;最后将rCSFV-Venus和表达EGFP的重组病毒rCSFV-EGFP以相同剂量感染PK-15细胞,通过流式细胞仪分析荧光强度及感染效率。结果表明:rCSFV-Venus全长感染性克隆构建成功;转染后传至第4代可观察到绿色荧光,表明重组病毒拯救成功;将拯救的病毒连续传代10代,每代感染的PK-15细胞都可观察到绿色荧光,表明重组病毒具有良好的稳定性;流式细胞仪分析结果表明,rCSFV-Venus比rCSFV-EGFP荧光强度强且感染效率高。说明本研究成功拯救了稳定表达Venus基因的rCSFV-Venus,为筛选介导CSFV入侵的功能性受体提供了有力工具。
In order to screen the functional receptor(s)mediating the entry of classical swine fever virus(CSFV)in combination with the CRISPR/Cas9 technique,the experiment first used fusion PCR technology to fuse the venus gene into amino acids between the 13th and 14th sites of Npro protein to obtain full-length infectious clone pCSFV-Venus.pCSFV-Venus was then transferred into SK6 cells to rescue recombinant rCSFV-Venus,and the rescued virus infected porcine kidney cells(PK-15 cells)for continuous passage to observe its stability.Finally,the rCSFV-Venus and the recombinant virus rCSFV-EGFP expressing EGFP were infected with PK-15 cells at the same dose,then fluorescence intensity and infection efficiency were analyzed by flow cytometry.The results showed that the full-length infectious clone of rCSFV-Venus was successfully constructed.Green fluorescence was observed at the fourth generation,indicating that the recombinant virus was successfully rescued.Furthermore,the green fluorescence was observed in each generation of infected PK-15 cells for 10 generations,indicating that the recombinant virus had good stability.Flow cytometry analysis showed that rCSFV-Venus had higher fluorescence intensity and infection efficiency than rCSFV-EGFP.The above results showed that this study successfully saved rCSFV-Venus with stable expression of Venus gene and provided a powerful tool for screening the functional receptor(s)mediating CSFV entry.
作者
孙慧敏
仇华吉
李素
李永锋
SUN Huimin;QIU Huaji;LI Su;LI Yongfeng(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2020年第20期67-71,80,158,159,共8页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31630080,31772774,31672537)。
关键词
猪瘟病毒
Venus基因
重组病毒
稳定性
荧光强度
感染效率
Classical swine fever virusqtne
venus gene
recombinant virus
stability
fluorescence intensity
infection efficiency
