稳定表达犬瘟热病毒Nectin4受体的Vero细胞系构建

doi:10.16431/j.cnki.1671-7236.2020.10.033

Abstract

Abstract: 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,Ig κ signal 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 6 μg/mL.Vero cell transfected with pCI-N4 plasmid would form significant cell clusters in presense of 6 μg/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.

Key words: canine distemper virus (CDV); Nectin4; receptor; Vero cell line

CLC Number:

S855.3

YU Zekun, DUAN Xiaoxiao, ZHI Yong, LUAN Zhifang, SHAN Xueqiang. Construction of Vero Cell Line Stably Expressing Nectin4 Receptor of Canine Distemper Virus[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3334-3342.

References 26

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

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