Isolation And Identification Of Porcine Rotavirus And Generation Of Monoclonal Antibodies In Southwestern Henan

Porcine Rotaviruses(PoRV)is major cause of the main pathogens of viral diarrhea in piglets,mainly causing vomiting,diarrhea and anorexia in young piglets.The disease is widespread and has a high incidence,causing serious economic losses to the livestock industry.To understand the genetic variation and epidemiological characteristics of Po RV in southwest Henan,this study used RT-PCR to perform pathogenic tests on clinical tissues and fecal samples sent from pig farms in southwest Henan in recent years to clarify its epidemiological trends and occurrence patterns.The Po RV vp6 gene fragment was further amplified to understand the variation information of Po RV strain,and further isolated and cultured in MA-104 cells for the detected strain to isolate the rotavirus strain.Subsequently,the prokaryotic expression vector of Po RV VP6 was further constructed,the purified protein was expressed and immunized in BALB/c mice,and the monoclonal antibody that could effectively bind VP6 was screened,which has high application value and is conducive to an in-depth understanding of the variation and prevalence of PoRV and provides biological materials for establishing a sensitive,rapid and simple Po RV detection method at a later stage.The conclusions are as follows:1.RT-PCR was used for epidemiological investigation and pathogen detection of diarrheal disease materials sent to the southwest of Henan from 2020 to 2022.The results showed that 54 positives samples were detected out of 230 sent diarrheal disease materials,with a total positivity rate of 23.48%.The highest incidence rate was 31.36%(37/118)in2021,and the survey of Po RV in different quarters revealed the highest positivity rate of31.25%(15/48)in the fourth quarter(October to December).Porcine rotavirus is prevalent mainly prevalent in October-December,in cold climates.2.Positive samples were tested by RT-PCR and genetic sequencing,and the vp6 gene sequences were subjected to genetic evolutionary analysis,and the amplified virulent strain vp6 was found to be predominantly type I5.Sequence alignment analysis revealed that 5strains were similar to FX17 strain,4 strains were similar to HB-KS96 strain,and 1 strain was similar to FNCY strain.10 isolates had 87.8%-99.6% nucleotide similarity and92.1%-99.4% amino acid similarity,with small differences.Compared with the six strains published in recent years in the collected Gene Bank database,the nucleotide similarity ranged from 84.2% to 99.7% and the amino acid similarity ranged from 91.2% to 99.7%.The positive samples were treated and inoculated into MA104 cells,and it was found that the uninfected cells had a small number of dead cells after 48 h of spreading plate culture,and a large number of cells were still growing against the wall,and the cells gathered and pulled the net after 48 h of infection,and the wall was reduced,and the blind transmission of six generations all had cytopathic lesions,and a strain of porcine rotavirus was successfully isolated,which was named He NNY202001.The amino acid sequence of VP6 amplified from He NNY202001 isolate was compared with the sequence of popular virulent strains in recent years using MEGA 11 software,and the similarity was 88.4%～98.3%.The similarity with Bovine RV was 89.5%,with Human RV was 94.3%,and the highest similarity with porcine origin was 97.1%.3.We further subjected the isolated rotavirus HeNNY202001 strain 1194 bp of vp6 gene to the construction of a prokaryotic expression vector and constructed a recombinant expression bacterium p ET28a-Msy B-Po RV-VP6 with a protein band at 70 k Da after IPTG induction.Western blot results showed that the recombinant porcine rotavirus VP6 protein with his tag reacted specifically with 6*His antibody.BALB/c mice aged 6-8 weeks were immunized with the recombinant protein,and the antibody level of the mice was detected by ELISA,which increased with the number of immunizations.After boosting immunization,single mice were analyzed for antibody potency,and those with the highest potency were subjected to isolation of splenocytes and preparation of hybridoma cells.After three rounds of subcloning,five positive hybridoma cell lines were finally screened.SDS-PAGE electrophoresis showed that the purified antibody had two bands,a heavy chain at 55 k Da and a light chain at 25 k Da.Western blot assay with the isolated Po RV identified2G9-11,4G1-8 and 4G2-8 monoclonal antibodies that specifically recognized the isolate HeNNY202001.