Preparation Monoclonal Anitibodies And Preliminary Screening Of Iteraction Proteins Of Porcine Deltacoronavirus N Protein

In 2012,porcine deltacoronavirus(PDCoV)was discovered from clinical samples by Hong Kong researcher Woo.PDCoV is an enteric pathogen of pigs,it can infect pigs of any age.Its clinical manifestations include vomiting,watery diarrhea,dehydration,and even death.The epidemic has caused huge losses to the large-scale pig industry in China.Because PDCoV is often mixed with other virus in the clinic,we need a fast and effective method for diagnosing PDCoV.In this study,the prokaryotic expression system was used to express and purify the soluble pET-32a-N recombinant protein.Three monoclonal antibodies against PDCoV N protein were successfully prepared.This study can provide a reliable basis for the analysis of the epitope and function of the PDCoV N protein,the establishment of a clinical diagnosis method for PDCoV,and the design and development of vaccines.At the same time,we used the monoclonal antibody against PDCoV N protein in co-immunoprecipitation,combined mass spectrometry and bioinformatics analysis,analyzed the interaction between the PDCoV N protein and TUBB2B.The specific research contents are as follows:1.Prokaryotic expression and identification of porcine delta coronavirus N protein.PDCoV N protein is a nucleocapsid protein encoded by the genome.It is the most stable protein after virus infection in host cells.N protein is an ideal target antigen for PDCoV detection.In view of this,we used the viral RNA of the PDCoV/SD2018/10 strain as a template,1026bp N gene was amplified by RT-PCR;a pET-32a prokaryotic expression vector carrying the PDCoV N gene was constructed and transformed into BL-21 competent cells;0.5mM IPTG is used to induce the expression of pET-32a-N recombinant protein;and verified by SDS-PAGE and Western blot.We successfully prepared the soluble pET-32a-N recombinant protein which is 56kDa,then it was purified by Ni column affinity chromatography.2.Preparation and identification of monoclonal antibodies against porcine delta coronavirus N protein.The purified pET-32a-N recombinant protein was used as an immunogen to immunize 6-week-old BALB/c female mice.The serum of immunized and blank mice was collected as positive and negative serum,and the pET-32a-N recombinant protein was established as an antigen.We used cell fusion and hybridoma cell screening techniques,three positive hybridoma cell lines capable of stably secreting anti-PDCoV N protein monoclonal antibodies were obtained,and were named 4D3,4E3,and 3B4.After indirect ELISA detection,the titers of ascites of the three monoclonal antibodies were all 1×10^-6,the heavy chains of the antibody subclasses were all IgG2a,and the light chains of the antibody subclasses were all Ig? chains.By Western blot and IFA identification,all three monoclonal antibodies can specifically bind the virus N protein of PDCoV/SD2018/10 strain.3.Preliminary identification of the interaction protein of porcine delta coronavirus N protein.In order to screen out the interacting proteins of PDCoV N protein,LLC-PK1 cells inoculated with PDCoV were used as the infection group,and LLC-PK1 cells not inoculated with the virus were used as the control group.The co-ip method was used to distinguish the differential protein bands between the infected and control groups.We got 68 potential interaction proteins of PDCoV N protein by mass spectrometry,and selected ANXA2?KRT8?SERPINH1 and TUBB2B as the candidate proteins by GO?COG?KEGG analysis.We finally confirmed that PDCoV N protein interacted with TUBB2B.