Study On D Antigen Of Poliovirus Type ? And Its Detection Kit

Polio is a major acute infectious disease caused by poliovirus(PV).In the last 50s,Salk inactivated vaccine and Sabin live vaccine were successfully developed,which effectively reduced the incidence rate of poliomyelitis.With the prevalence of polio under good control and approaching the eradication stage,the WHO proposed a vaccination strategy from OPV to IPV and recommended that vaccine manufacturers use Sabin strain with weak virulence to produce IPV vaccine,in order to achieve the goal of global polio eradication.PV has three serotypes,each of which is composed of two different virus particles:one is D antigen,which is an infectious complete virus particle;the other is C antigen,which is an immature hollow virus particle.The main effective component of IPV vaccine is D antigen.In this study,the differences between C antigen and D antigen of Sabin strain of poliovirus type ? in morphology,infection characteristics,nucleic acid content and structural gene nucleotide sequence,different structural protein content and immunogenicity of the two antigens were observed,and a double antibody sandwich ELISA system for quantitative detection of D antigen of poliovirus type ? was establishedAfter ultrafiltration concentration and cesium chloride density gradient centrifugation,the C antigen and D antigen were obtained from the inactivated production stock solution or non inactivated virus purification solution of poliovirus type ? Sabin strain.The transmission electron microscope showed that C antigen was empty shell,D antigen was solid shell,and there were mixed D antigen particles in C antigen population;The results of SDS-PAGE showed that the coat protein of C antigen was composed of VPO,VP1 and VP3,and the coat protein of D antigen was composed of VP1,VP2,VP3 and VP4.The VP1 content of D antigen with the same quality was nearly 1.7 times as that of C antigen,the VP3 content of C antigen was nearly three times that of D antigen;The purity of C antigen can be improved by reducing the concentration of sample in ultracentrifugation;The purity of C antigen can be also improved after twice ultracentrifugation.Virus titers and nucleic acids of two batches of inactivated C and D antigens separated by two-step ultracentrifugation were detected.The results showed the virus titer of unit mass D antigen was about 4 logarithms higher than that of C antigen.This result is similar to that of electron microscopy,in which two suspected D antigen particles can be found in nearly ten thousand C antigen populations.Quantitative RT-PCR was used to detect the nucleic acid content of C antigen and D antigen.It was found that the nucleic acid content of each microgram of C antigen amplified by RT-PCR was equivalent to that of 1/64 microgram of D antigen amplified.Therefore,the effective nucleic acid template of VP1 gene of C antigen was only 1/64 of D antigen.The results of C antigen and D antigen structure gene sequencing showed that.The nucleotide sequences of C antigen and D antigen were identical.The results of immunogenicity test of C antigen and D antigen showed that in the first week of primary immunization,the antibody reached the peak value and the neutralizing antibody induced by C antigen is only 1/10-1/50 of D antigen.After the second immunization with C antigen and D antigen,the peak value of antibody usually reached in one to two weeks,which was 10-40 times higher than that before immunization.The neutralizing antibody induced by the same dose of D antigen is 20-40 times higher than that of C antigen,which shows that D antigen plays an important role in the effectiveness of IPV vaccine.Rabbit and goat were immunized with inactivated sPV ? D antigen to prepare polyclonal serum.Purification of polyantic serum by caprylic acid-ammonium sulfate precipitation method,and the purified polyclonal serum was labeled with HRP by sodium periodate method.The detection kits assembled with different purified antiserum can detect sPV ? virus in the range of 2-16 ?g/mL.The same concentrations of D antigen and C antigen were tested by different kits.The results showed that the binding D antigen was much higher than C antigen,and the detection difference was more than 2.0 OD values.The correlation coefficient between the concentration of D antigen and the corresponding OD value detected by the kits was greater than 0.99.A double antibody sandwich ELISA system was established with sheep polyclonal antibody as capture antibody and 3 × rabbit polyclonal antibody HRP as detection antibody.The linear correlation coefficient of this sandwich combination is R2>0.99,the linear detection range is 0.25-8 DU/ML,the specificity is good,and there is no cross reaction with antigens other than sPV ? D antigen.The above results provide experimental basis for the detection and identification of polio vaccine C and D antigens and further study of vaccine efficacy.