Preparation Of Monoclonal Antibodies Targeting ASFV P30 And SARS-CoV-2 N

African Swine Fever(ASF)is a highly contagious and lethal disease of pigs caused by African Swine Fever Virus(ASFV).ASF has endangered the global swine industry and resulted in serious economic losses to pig-producing countries.At present,a substantial breakthrough has not yet made in the research and development of the ASF vaccine.Rapid and sensitive diagnostic assays are necessarily required for the early detection of ASF epidemics and the implementation of biosafety measures.The ASFV genome is large,encoding nearly 200 kinds of proteins,and the structure of virus particle is complex.Among them,p30 is an important candidate protein for the development of detection technology since it could induce antibody production in the early stage of infection and demonstrated highly potent immunogenicity.At the same time,the emerging novel coronavirus pneumonia(Corona Virus Disease 2019,COVID-19)also poses a huge threat to the public health,which is an acute and severe infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)with cough,fever,and dyspnea as the main symptoms.Recently,accurate detection is still an effective strategy to prevent and control the spread of SARS-CoV-2.The nucleocapsid(N)protein is the most abundant structural protein of SARS-CoV-2,which expresses at the initial stage of infection and participates in RNA packaging.It can be used as an important research target for COVID-19 diagnosis and therapeutic drug development.Therefore,the stable and specific monoclonal antibodies(mAb)against ASFV p30 and SARS-CoV-2 N proteins were prepared in this study,respectively.The main contents are as follows:1.Preparation of targeting ASFV p30 mAbIn this experiment,the pET-32a-p30 prokaryotic expression vector was constructed and induced,and the inclusion bodies formed by the p30 recombinant protein were purified by nickel column chromatography.The female BALB/C mice aged 6-8 weeks were immunized with 200 μg of purified recombinant protein,the spleen cells were collected and then fuse with SP2/0 myeloma cells.After three rounds of subcloning,a hybrid strain secreting specific targeting p30 antibody is obtained,named 8H9.Western blot analysis of showed that the prepared mAb displayed good reactivity to p30 recombinant protein and ASFV,respectively.The result of indirect immunofluorescence assay(IFA)further indicated that the mAb can specifically recognize the viral antigen in ASFV-infected cells.The subclass of the prepared mAb belonged to Ig G1/κ.2.Preparation of SARS-CoV-2 N mAbUsing the same preparation strategy as the ASFV p30 mAb,two strain hybridoma secreting SARS-CoV-2 N-specific mAbs were successfully screened in this experiment,designated 2D11 and 8G6.Western blot analysis demonstrated that these mAbs has good reactivity to N recombinant protein.IFA analysis of HEK-293 T transfected with eukaryotic expression plasmid pc DNA6B-n CoV-N-Flag confirmed that the generated mAbs presented the specific response to SARS-CoV-2 N protein.Both of these prepared mAbs were Ig G1/κ subclassThe mAbs binding to ASFV p30 and SARS-CoV-2 N prepared in this study will be conducive to explore the role of the above proteins in viral infection,and promote the development of rapid diagnostic technology and vaccine.