Screening Of Monoclonalantibodies With Blocking Activity Against Cat IL-1β And Cat Origin Modification

Interleukin-1(interleukin-1,IL-1),referred to as IL-1,is an important regulator of immune and inflammatory response.IL-1β is the main member of the biological function.It is widely involved in inflammation,tissue destruction,edema formation and other pathological injury processes.A large amount of release of IL-1β can be found in hosts infected with viruses,bacteria,fungi and parasites.Therefore,IL-1βhas become an important target for the treatment of inflammatory diseases.The blocking monoclonal antibody against IL-1β has become an effective drug for the treatment of inflammatory diseases because it can bind to IL-1β with high affinity and block its interaction with receptors and activation of downstream signal pathways.In recent years,with the increase of companion animals such as cats and dogs,people pay more and more attention to the prevention and treatment of pet diseases.IL-1β is also related to a variety of inflammatory diseases in cats,such as glomerulonephritis,gastroenteritis,esophagitis and so on.In order to fill the vacancy of cat IL-1βblocking activity monoclonal antibody in the market,on the basis of expressing and purifying the bioactive cat IL-1β recombinant protein,the cat IL-1β monoclonal antibody with blocking activity was screened and modified,so as to provide a new product for the treatment of cat inflammatory diseases.The details are as follows:1.Expression,purification and bioactivity detection of cat IL-1β protein.The total RNA of cat spleen tissue was used as template to amplify cat IL-1βgene by RT-PCR.The amplified product was cloned into prokaryotic expression vector to construct recombinant plasmid p ET30a-IL1β.The recombinant plasmid was transferred into E.coli expression system,and the target protein was proved to be expressed in the supernatant of bacteria by Western Blot and indirect immunofluorescence experiments.Then the Escherichia coli expressing IL-1β was cultured in large quantities,and the protein was purified by nickel column affinity chromatography,and the pure cat IL-1β protein was obtained by SDS-PAGE and Western Blot experiments.The purified IL-1β recombinant protein was transfected into HEK-293 T cells with NF-κ B luciferase reporter system.It was proved that the protein could activate the transcription factor activity of NF-κ B.At the same time,the expression of inflammatory factors in F81 cells treated with feline IL-1βrecombinant protein was detected by fluorescence quantitative RT-PCR,which proved that the protein could induce the expression of inflammatory cytokines in F81 cells,indicating that the purified cat IL-1β recombinant protein had biological activity.2.Screening of cat IL-1β monoclonal antibodies with blocking activity.Six-week-old female BALB/C mice were immunized by subcutaneous injection of purified and verified cat IL-1β protein with Freund adjuvant.Blood was collected from the submandibular vein of mice after three immunizations.The antibody titer of cat IL-1β protein in serum was detected by ELISA,and the mice with the highest antibody titer were selected for impact immunization.after 7 days of impact immunization,the spleen of mice was taken to prepare immune splenocytes.And fused with SP2/0 myeloma cells.After repeated subcloning for three times,498 hybridoma cells with high OD value were screened.Through NF-κ B luciferase reporting system,the supernatant of two monoclonal cells showed cat IL-1β blocking activity,which were named H10 E and C4 C respectively.Western Blot and indirect immunofluorescence experiments showed that the supernatants of H10 E and C4 C and other 6 monoclonal cells with high OD value could react with cat IL-1β protein.After immunizing the mice with the 8 monoclonal cells,the ascites was collected and the antibody was purified.The purified H10 E and C4 C Mc Abs were co-incubated with IL-1β protein at different concentrations,and their blocking activity was verified by NF-κ B luciferase reporter system and fluorescence quantitative RT-PCR.The results showed that two monoclonal antibodies with blocking activity were successfully obtained.3.Analysis of the action sites of monoclonal antibodies against IL-1βblocking activity in cats.According to the prediction results of cat IL-1β functional site by Shengxin software,it was truncated and expressed in three segments,named N1,N2 and N3,respectively.Using the amplified cat IL-1β gene as template,three gene fragments were amplified by PCR and cloned into prokaryotic expression vector p ET-30 a to construct prokaryotic expression plasmids p ET-30a-IL1β-N1,p ET-30a-IL1β-N2 and p ET-30a-IL1β-N3.After induced expression and purification,IL1β-N1,N2 and N3 proteins were obtained.Two monoclonal antibodies with blocking activity were reacted with three recombinant proteins respectively.Western Blot experiments showed that monoclonal antibody H10 E targeted IL-1β N3 region and monoclonal antibody C4 C targeted IL-1β N2 region.4.Cat Origin Modification of Monoclonal Antibodies against Cat IL-1βblocking activity.The total RNA of H10 E and C4 C monoclonal cells was extracted,and the variable region sequences VH and VL of the antibody were amplified by RT-PCR and sequenced.They were connected to the eukaryotic plasmid containing the constant region of cat antibody and transfected into HEK-293 T cells.After verifying the successful expression,the supernatant of the cell culture medium was collected and concentrated,and the concentrated solution was incubated with IL-1β protein.Through the detection of NF-κ B luciferase report system and the expression level of inflammatory factors,it was proved that the cat-derived modified antibody could block IL-1β.To sum up,the recombinant cat IL-1β protein with biological activity was successfully prepared in this study,and two cat IL-1β monoclonal antibodies with blocking activity were screened.After cat source modification,they have the potential to be developed as blocking activity monoclonal antibody drugs against cat IL-1β.