Isolation And Identification Of Porcine Deltacoronavirus From Xi'an Wild Strain And Immunogenicity Of M Protein

Porcine deltacoronavirus?PDCoV?is a newly discovered enteric coronavirus that can cause diarrhea,vomiting,dehydration,and death in newborn piglets.The fatality rate can reach30%to 40%,seriously reduced the survival rate of newborn piglets,affected the development of the pig industry.Several provinces in China have isolated PDCoV local strains,but to date no reports of PDCoV Shaanxi local strains have been isolated.The successful isolation of PDCoV local strain is of great significance for the pathogenesis of PDCoV,virological and serological analysis,and vaccine development in Shaanxi.The structural protein expressing PDCoV provides the basis for the subsequent development of diagnostic reagents and potential PDCoV subunit vaccines.This experiment collected the intestines and contents of piglets with diarrhea from a pig farm in Xi'an,Shaanxi Province.Swine testis?ST?cells were used to isolate pathogens.When the cells showed significant lesions,the full sequence of M gene of PDCoV Xi'an strain was amplified by RT-PCR.Sequencing to explore whether the B cell epitope has changed.The M gene and the expression vector p ET-32a were used to construct recombinant plasmids,which were transferred into BL21?DE3?strains to induce expression.The purified recombinant protein was used to immunize mice to prepare multiclonal antibodies.This study achieved the following results:1.PDCoV isolates were passed on blindly for 5 generations.Cytopathic lesions appeared72 hours after infection with ST cells,which were mainly manifested as rounding,enlargement,aggregation and shedding of cells.RT-PCR showed that PDCoV was positive and named CHN-XA18-35 strain.TCID₅₀of F5 generation cytotoxicity in ST cells is 10^-5.81/100?L.2.Sequencing results showed that the M gene of this isolate had a nucleotide similarity of 98.47%to 99.54%and a similarity of amino acids of 97.24%to 99.08%with reference strains in other regions.The amino acid position 189 of the M protein was mutated from isoleucine?Ile?to arginine?Arg?.3.A M-1-pET-32a recombinant expression vector was constructed,the optimized expression conditions were an induction temperature of 25?,an inducer concentration of 0.5mmol/L,and an induction time of 20 h.The expressed recombinant protein has a molecular weight of approximately 34 ku.4.Antiserum was prepared by immunizing mice with the purified recombinant protein,and the neutralizing antibody titer was 1:179.Can be used as an indirect immunofluorescence method to detect Porcine deltacoronavirus.In conclusion:1.The PDCoV Shaanxi local strain was successfully isolated and identified.The mutation of amino acid 189 of M protein has no effect on its B cell epitope.2.The recombinant protein has good immunogenicity,and PDCoV-M antiserum has a good neutralizing effect.