Preparation And Detection Of Monoclonal Antibodies Against Chicken Parvovirus NS1 And VP2 Proteins

Chicken parvovirus(ChPV)is an important pathogen causing developmental retardation and Runting stunting syndrome(RSS)in chickens,mainly infecting low-day-old broilers.It leads to developmental retardation,diarrhea,immune dysfunction and decreased feed conversion rate of infected chickens,resulting in significant economic losses to the poultry industry.Currently,functional studies of NS1 and VP2 proteins during ChPV infection are limited due to the lack of specific antibody detection tools and the lack of clinical serological test kits that can rapidly screen for ChPV.Therefore,it is particularly important and urgent to develop and prepare monoclonal antibodies against non-structural protein NS1 and structural protein VP2 for rapid detection of ChPV.In this study,NS1 and VP2 gene sequences of GX-Ch-PV-21 strain(Gen Bank entry number:MG602511)were synthesized artificially,and the specific primers were designed to amplify the NS1 and VP2 genes.The amplified products were connected to the p ET-32a(+)expression vector,respectively,and the recombinant plasmoids p ET-32a-NS1 and p ET-32a-VP2were obtained successfully.The two recombinant plasmogenes were transferred into thansetta(DE3)receptor cells,and the expression products were analyzed by SDS-PAGE electrophoresis after the induction conditions were optimized.The recombinant proteins of NS1 and VP2 were well expressed.Soluble analysis showed that the two recombinant proteins mainly existed in the form of inclusion bodies.The results of SDS-PAGE showed that the purification effect of the two purified proteins was good.Western-Blot results showed that the two recombinant proteins could bind specifically to the anti-HIS antibody,indicating that the protein expression was correct.Polyclonal antibodies were prepared by immunizing SPF chickens with two recombinant proteins,respectively.Western-Blot results showed that VP2 and NS1 polyclonal antibodies could bind specifically to corresponding proteins.IFA assay results showed that both polyclonal antibodies could bind specifically to corresponding recombinant proteins.This study lays a good foundation for the further development of ChPV detection methods.Balb/c mice were immunized with two recombinant proteins,and after cell fusion and indirect ELISA screening,two hybridoma cell lines targeting NS1 were obtained,named 1B12 and 2B2,and one hybridoma cell line targeting VP2 was obtained,named 3F8.The titers of 1B12,2B2 and 3F8 were1.6×10~7,4.1×10~6and 8.2×10~6,respectively.The identification results showed that the heavy chain of 1B12 and 2B2 was Ig G1,the heavy chain of 3F8 was Ig G2a,and the light chain of the three monoclonal antibodies was kappa chain.The results of Western-Blot and IFA showed that the monoclonal antibody had good specificity.The monoclonal antibody prepared in this study lays a material foundation for the establishment of double antibody sandwich ELISA method.In order to establish a detection method for ChPV antigen,two sandwich ELISA methods,NS1-DAS-ELISA and VP2-DAS-ELISA,were established by using monoclonal antibody and polyclonal antibody prepared by non-structural protein NS1 and structural protein VP2 of ChPV.TaqMan probe qPCR method was established based on NS1 gene sequence.The experimental results showed that the three methods had strong specificity,high sensitivity and good repeatability.The results of NS1-DAS-ELISA method,VP2-DAS-ELISA method and TaqMan probe qPCR method established in this study were compared with the results of nested PCR method that has been used in clinic,and the coincidence rates were 89.1%,81.3%and 96.9%,respectively.All the three methods can be used for the detection of clinical samples of ChPV.In conclusion,in this study,NS1 and VP2 proteins of ChPV were expressed prokaryotically,and the recombinant protein was used as the antigen to prepare polyclonal antibodies and monoclonal antibodies against NS1 and VP2,respectively,to provide material for the research and development of ChPV diagnostic technology.A sandwich ELISA assay based on two proteins and a TaqMan probe real-time quantitative PCR assay based on NS1 gene sequence were established to provide technical support for the prevention and control and diagnosis of ChPV.