稳定表达犬瘟热病毒Nectin4受体的Vero细胞系构建

Construction of Vero Cell Line Stably Expressing Nectin4 Receptor of Canine Distemper Virus

为提高病毒的分离效率,本试验设计构建了能够表达犬瘟热病毒(canine distemper virus,CDV)上皮细胞Nectin4受体的Vero细胞。为增加蛋白定位的准确性并易于鉴定,使用Igκ信号肽替换原有信号肽序列并添加了HA标签,在Nectin4 ORF后串联IRES-Puro序列并连入pCI-Neo真核表达载体,得到完整的转染载体pCI-N4。不同浓度嘌呤霉素孵育Vero细胞得到最小筛选浓度为6μg/mL。pCI-N4重组质粒转染Vero细胞后使用6μg/mL嘌呤霉素筛选,5~7 d后出现具有抗性的细胞簇,有限稀释法连续单克隆纯化3代后获得稳定表达的细胞系。构建细胞系传代至15代能检测到Nectin4 mRNA转录,Western blotting检测筛选细胞得到约60 ku目的蛋白表达,间接免疫荧光检测显示纯化细胞蛋白表达丰度高且表达均一,激光共聚焦观察Nectin4目的蛋白定位于细胞膜,说明筛选的Vero-Nectin4细胞系能够稳定表达,表达蛋白能够满足作为CDV受体的结构要求。临床CDV阳性病料经研磨滤菌处理后接种构建细胞系能够产生典型的合胞体细胞病变,Vero对照组盲传3次未有病变。分离毒株TICD50=10-5.9/0.1 mL。构建的Vero-Nectin4细胞系可用于CDV分离。

In order to improve the efficiency of virus isolation,Vero cells were designed and constructed to express the epithelial cell receptor of canine distemper virus(CDV).In order to improve the accuracy of protein localization and easy identification,Igκsignal peptide was used to replace the original signal peptide sequence and HA tag was added.IRES-Puro sequence was concatenated with Nectin4 ORF and inserted into pCI-Neo eukaryotic expression vector to obtain the complete transfection vector pCI-N4.The minimum dose of puromycin to kill all monolayer Vero cell was 6μg/mL.Vero cell transfected with pCI-N4 plasmid would form significant cell clusters in presense of 6μg/mL puromycin after 5-7 d.The stable cell line expressing Nectin4 receptor was obtained by 3 times limiting dilution method.The F15 cell could still detect Nectin4 mRNA transcription and almost 60 ku interest protein by Western blotting.By indirect immunofluorescence,the Nectin4 protein expressed abundantly and equably.Cellular localization of Nectin4 protein was on the cytomembrane in the laser confocal microscope field.The Nectin4 protein correctly expressing on membrane met the structural requirement as viral receptor.The stable cell line incubated with a canine distemper positive asepsis simple generated obvious syncytium CPE.And ordinary Vero had no CPE after 3 times blind passage.TCID50 of isolated wild-type strain was 10-5.9/0.1 mL.In conclusion,the Vero cell which expressed the modified Nectin4 receptor stably was available for CDV isolation.