Isolation And Identification Of PEDV Sichuan Strain (CHN-SC2021) And Preparation Of Monoclonal Antibody Against N Protein

Porcine epidemic diarrhea（PED）mainly causes diarrhea and death in piglets,causing huge economic losses to the pig industry.After 2010,the prevalence of PEDV variants has further led to increased difficulties in the prevention and control of PED in China.In this study,a PEDV epidemic strain was isolated from pig farm samples with diarrhea outbreaks in Sichuan.The culture characteristics,genome wide characteristics,and pathogenicity of the strain were systematically identified,and monoclonal antibodies against N-protein were prepared,providing more reference information for the research and prevention and control of PEDV in China.The main contents of this study are as follows:1.Isolation and identification of PEDV Sichuan strain CHN-SC2021The diarrhea material identified as PEDV positive by RT-PCR was inoculated with Vero cells for continuous passage,and the PEDV virus strain was isolated.The isolated PEDV strain was identified by plaque purification,indirect immunofluorescence assay（IFA）,and negative staining transmission electron microscopy.The strain was named CHN-SC2021.The pathogenic characteristics of the virus strain were dynamically monitored.The virus strain was continuously subcultured on the cell for 35 generations,and the time of obvious cytopathic effect in the cell changed from 48 h to 24 h.The titer increased from 10^4.7 TCID₅₀/m L to 10^6.43TCID₅₀/m L.The growth kinetics analysis of the P35 generation showed that the virus replication reached a peak at 48 hours.The whole genome sequencing showed that the total length of CHN-SC2021（P5）（Genbank accession number:OM505025.1）was 28014 bp.Compared with major reference strains worldwide,it was found that CHN-SC2021 had 99.16～96.32%,99.01～96.57%,and 98.94～96.33%homology with Chinese strains,European and American strains,and strains from other Asian countries,respectively;The results of genetic evolution tree showed that CHN-SC2021 strain was closely related to Jiangxi CH/JXJA/2017（MF375374.1）,Henan CH/HNAY/2015（KT199103.1）,and CH/HNKF-16（KY649107.1）strains,belonging to GⅡa virus strain.The similarity of S gene with other strains is between 98.7%and 93.4%,and it is similar to Korean strain KNU1305（KJ662670.1）,American strain IA2（KF468754.1）,and USA strain＿Colorado＿2013（KF272920.1）is closely related and no potential restructuring events have been identified.Oral administration of CHN-SC2021 to 6-day old sucking piglets resulted in loss of appetite at 12 h,vomiting at 20 h,and anorexia,severe vomiting,and diarrhea in piglets at 24 h;The main pathological changes on autopsy were gastrointestinal distension,transparent jejunum and ileum;Pathological changes showed varying degrees of intestinal villi lesion,desquamation,loose connective tissue,and widening of the muscularis mucosa to muscularis space in each intestinal segment;Immunohistochemistry showed that viruses were present in all segments of the small intestine,with the largest number of viruses in the ileum.2.CHN-SC2021 N-protein expression and preparation of monoclonal antibodyIn this study,the full-length N gene of CHN-SC2021 strain was amplified,and a recombinant plasmid p ET-28a-N was constructed by inserting a p ET-28a（+）vector.An N protein with a size of about 60 k Da was obtained through IPTG induction and expression.The expressed protein mainly existed in bacterial supernatant.An indirect ELISA method was constructed using purified recombinant N protein,and the optimal coating concentration of N protein antigen was 1μg/well.Balb/c mice were immunized with N protein,and their spleen cells were fused with SP 2/0 cells.A hybridoma cell line 8F12 with the best immunogenicity was selected to prepare monoclonal antibodies.8 weeks old female Balb/c mice were injected intraperitoneally to prepare ascites.The ascites were purified using octanoic acid saturated ammonium sulfate method and Protein G purification column to obtain a 1:102400 titer monoclonal antibody.The identification results showed that the heavy chain of the monoclonal antibody belonged to Ig G1 type,and the light chain belonged to Kappa type,The prepared monoclonal antibody can specifically recognize PEDV-N protein and has no cross reaction with TGEV and PDCo V.The monoclonal antibodies react specifically with PEDV viruses in vitro cells and specifically bind to viruses in the duodenum,jejunum,and ileum of pigs infected with PEDV.The monoclonal antibody against N protein prepared in this study provides scientific research material for subsequent studies such as the identification and diagnosis of PEDV.