Preparation And Preliminary Application Of CHI3L1 Polyclonal Antibody

The inflammatory response induced by influenza virus infection is the main cause of death in animals.As an inflammatory marker,the detection of CHI3L1 in animal serum can provide a theoretical basis for the judgment of the process of animal inflammatory response,and provide support for the subsequent detection and diagnosis of animal diseases.The aim of this study was to establish a diagnostic method for the progression of inflammatory response by detecting CHI3L1,an inflammatory marker.It is of great significance for the subsequent improvement and treatment of inflammation.The main results of this experiment are as follows:1.Validation of the relationship between CHI3L1 and inflammatory response induced by influenza virus.In this study,the CHI3L1 expression level was detected by in vitro and in vivo infection test,the CHI3L1 level in mice infected with H1N1 was detected by ELISA and IF staining,and the change of CHI3L1 expression level in A549 cells infected with H1N1 was detected by q PCR and Western blot.The results showed that CHI3L1 was significantly increased in both in vivo and in vitro.CHI3L1 was positively correlated with the degree of influenza virus infection.On this basis,we will prepare CHI3L1 polyclonal antibody and establish an ELISA assay to diagnose the severity of inflammatory response induced by influenza virus infection in animals.2.Prokaryotic expression and purification of CHI3L1.CHI3L1 primers were found and designed by NCBI.After PCR amplification,they were connected to the expression vector p ET-28 a through homologous recombination.The recombinant plasmid p ET-CHI3L1 was obtained and identified by colony PCR.The identified p ET-CHI3L1 recombinant plasmid was transformed into the receptor state Rosetta for IPTG induction.The induction conditions showed that the protein was expressed as inclusion body,so it was denatured after inclusion body induction.8 M saturated urea solution was used for denaturation.The denatured solution was purified using his-tag affinity media.After purification,the protein was regenerated by dialysis using gradient urea solution.The target protein was obtained and verified by Western blot.3.Preparation and purification of CHI3L1 polyclonal antibody.Immunize rabbits with the CHI3L1 protein obtained for five times,then collect serum to identify CHI3L1 antibody and determine antibody titer by Western blot.Western blot results showed the presence of CHI3L1 antibody in rabbit serum,and ELISA was used to detect the antibody titer reaching 1: 6400,was purified using CNBr-activated Bestarose 4FF affinity medium conjugated to CHI3L1 protein.The CHI3L1 polyclonal antibody was determined by SDS-PAGE and ELISA.4.Establishment and application of indirect ELISA detection method.Indirect ELISA detection method and bispecific antibody sandwich colloidal gold detection method were established using purified CHI3L1 polyclonal antibody.The results of optimization of indirect ELISA conditions were as follows: the optimal antigen coating condition was 400 ng/μL,incubated at 37°C for 2 h.The optimal blocking conditions were 2% BSA,incubated at 37 °C for 2 h;The optimal primary antibody conditions were 1:200 dilution and incubation at 37°C for 1 h;The optimal secondary antibody conditions were 1:4000 dilution and incubation at 37°C for 2 h;The optimal color rendering condition was 15 minutes at room temperature.The results showed that this method could detect a significant increase in CHI3L1 protein(P<0.05)in the serum of H1N1 mice and Newcastle disease virus poultry,and also detected a significant increase in CHI3L1 protein expression in CHI3L1 stable cell line.In summary,by exploring the relationship between CHI3L1 and influenza virus-induced inflammatory response,prokaryotic expression of CHI3L1 protein and preparation of polyclonal antibodies,CHI3L1 indirect ELISA detection method was established,which can be used to detect CHI3L1 protein levels in animal serum,and indirect ELISA methods can also be used to detect CHI3L1 protein levels in cells.The results provide a theoretical basis for exploring the interaction between influenza virus and CHI3L1,and also provide a detection method for the diagnosis of viral disease infection.