摘要
猫氨基肽酶N(fAPN)参与猫传染性腹膜炎病毒(FIPV)的感染过程,为研究fAPN对FIPV复制的影响,本研究针对fAPN第14外显子设计了1对sgRNA,将sgRNA退火后克隆至pX458质粒中,构建同时含有Cas9蛋白和sgRNA的重组质粒.重组质粒经PCR及测序鉴定正确后,瞬时转染猫肾细胞(CRFK)中,24 h后通过流式细胞仪分选带有绿色荧光的单克隆细胞.单克隆细胞经稳定传代培养后,细胞中的绿色荧光消失,对细胞进行PCR和测序,结果显示:3株单克隆细胞在fAPN第14外显子有1个碱基的插入,造成移码突变,导致fAPN不能正常表达,表明敲除fAPN蛋白的CRFK细胞系正确构建,命名为KO-APN14.将FIPV分别感染CRFK细胞及培养20代的KO-APN14细胞,48 h后通过间接免疫荧光试验(IFA)检测FIPV N蛋白的表达;将FIPV分别感染CRFK细胞及KO-APN14细胞,在感染4h、12 h、24 h、36 h时采用RT-qPCR分别检测FIPV N基因、视黄酸(维甲酸)诱导基因Ⅰ(RIG-Ⅰ)、黑色素瘤分化相关基因5(MDA5)、Toll样受体3(TLR3)、干扰素β(IFN-β)、肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)的转录水平.IFA结果显示:感染FIPV的CRFK细胞出现绿色荧光,未感染FIPV的CRFK细胞、未感染FIPV的KO-APN14细胞及感染FIPV的KO-APN14细胞均无绿色荧光;RT-qPCR检测FIPV感染各细胞后病毒N基因的转录水平,结果显示:与感染FIPV的CRFK细胞相比,感染FIPV的KO-APN14细胞能够极显著抑制FIPVN基因的转录(P＜0.0001);RT-qPCR检测FIPV感染各细胞后上述各细胞因子的转录水平,结果显示:与感染FIPV的CRFK细胞相比,感染FIPV的KO-APN14细胞能够抑制RIG-Ⅰ、MDA5、TLR3、IFN-β、IL-6、IL-10、TNF-α及fAPN的转录.综上所述,fAPN是FIPV在侵入细胞及病毒复制过程中发挥重要作用,并在FIPV引起的炎症反应过程中起关键作用.本研究为fAPN在免疫炎症反应中作用的研究提供科学依据,并为培育基因编辑抗病育种猫奠定实验基础.
Abstract
In order to study the impact of feline aminopeptidase N(fAPN)on feline infectious peritonitis virus(FIPV)replication,a pair of sgRNA was designed for the fAPN exon 14,and the sgRNA was annealed and cloned into the pX458 plasmid to construct a recombinant plasmid containing both Cas9 gene and sgRNA.After identification by PCR and gene sequencing,the recombinant plasmid was transiently transfected into cat kidney cells(CRFK).After 24 hours of culture,monoclonal cells with green fluorescence were sorted by flow cytometry.After stable cell subculture of the monoclonal cells,the green fluorescence of the cells disappeared.The results of PCR and gene sequencing of the cells showed that three monoclonal cells line had one base insertion in the fAPN exon 14,causing a frameshift mutations,resulting in the abnormal expression offAPN.These results showed that the CRFK cell line with knockout of fAPN protein was successfully constructed and we named this cell line as KO-APN14.CRFK cells and KO-APN14 cells after 20 passages were infected with FIPV.After 48 hours,FIPV N protein expression was detected by indirect immunofluorescence assay(IFA).RT-qPCR was used to detect transcription levels of FIPV N gene,retinoic acid(tretinoin)-induced gene protein Ⅰ(RIG-Ⅰ),Melanoma differentiation-related gene 5(MDA5),Toll-like receptor 3(TLR3),interferon β(IFN-β),tumor necrosis factor α(TNF-α),interleukin-6(IL-6),and interleukin-10(IL-10)at 4 hours,12 hours,24 hours,and 36 hours post infection.The results of IFA showed that CRFK cells infected with FIPV emitted green fluorescence,while CRFK cells without FIPV infection,KO-APN14 without FIPV infection and KO-APN14 infected with FIPV did not have green fluorescence.RT-qPCR detected the transcription level of N gene after FIPV infection in each cell,and showed that KO-APN14 cells infected with FIPV significantly inhibited the transcription of FIPV N gene compared with CRFK cells infected with FIPV.RT-qPCR detection of the transcription level of the above cytokine showed that KO-APN14 cells infected with FIPV could inhibit the transcription of RIG-Ⅰ,MDA5,TLR3,IFN-β,IL-6,IL-10,TNF-α and fAPN compared with CRFK cells infected with FIPV.In summary,fAPN plays an important role in the process of FIPV invasion and virus replication,and is also an indispensable factor in the immune inflammatory response caused by FIPV.This study provides a scientific basis for the study of the role of fAPN in immune inflammatory response,and lays an experimental foundation for the breeding of gene-edited disease-resistant breeding cats.
基金项目
国家重点研发计划(2022YFF0710501)
中央级公益性科研院所基本科研业务费专项(1610302022018)
兽医生物技术国家重点实验室项目(SKLVBP202101)
兽医生物技术国家重点实验室项目(SKLVBP202120)
NETL
NSTL
万方数据