Establishment Of A Method For Detecting The Relative Content Difference Between Pseudorabies Virus And Rseudorabies Virus Antigen Before And After Purification

Pseudorabies virus(PRV)belongs to herpesvirus family a herpesvirus subfamily,varicella virus and porcine herpesvirus type I.PRV can cause pseudorabies.In China,the first report of pseudorabies in 1947 was that cats were infected with PRV,and then pigs,cattle,sheep and other animals were infected with PRV.So far,at least 18 provinces,cities,autonomous regions(including Taiwan)and Hong Kong autonomous region have the disease.PRV has no obvious seasonal epidemic.It can occur all year round,and its transmission mode is vertical transmission and media transmission.There are obvious neurological symptoms after PRV infection,which seriously invades the central nervous system.There are many hosts of PRV,animal,dog and cat,and wild animals are its hosts.Pigs are natural hosts and main sources of infection.Pseudorabies can hardly be controlled once swine outbreak is launched,and it can only eliminate the sick pigs and cause huge economic losses to the pig industry.Besides,the high incidence rate and high mortality rate of PRV also bring harm and loss to other aquaculture industries in China,and there are reports that PRV can infect humans.PRV is more and more harmful to human society.The prevention of pseudorabies caused by PRV has always been the focus of our country,and the detection of PRV is the premise of prevention.Traditional methods to detect PRV,such as pathogen isolation,take a long time and can not be detected in a wide range.At present,there is no effective drug to treat pseudorabies,which mainly depends on vaccine for prevention.However,the traditional inactivated vaccine contains too many impurities,which affects the effect of the vaccine,so the protein can be purified,that is,PRV can be purified.The glycoprotein of virus can stimulate the immune response of the body.It can be used as antigen to bind specifically with antibody.Therefore,a detection method can be established to detect PRV quickly and accurately,and the difference of antigen content of PRV before and after purification can also be compared.In this study,the pseudorabies virus was expanded and purified.The samples before and after purification were detected by TCID50,SDS-PAGE electrophoresis,Western blot,animal experiment and neutralization test to compare the changes of pseudorabies virus before and after purification,the most important of which was to observe the changes of protein.A double antibody sandwich ELISA method was also established in this experiment.Its principle is the specific combination of antigen and antibody.The working concentration,coating time,blocking time,selection of blocking solution,reaction time and color development time of capture antibody and enzyme labeled antibody were optimized.PRV was detected by this method,and the antigen content of PRV before and after purification was compared.In this study,pseudorabies virus was purified,and a double antibody sandwich ELISA was established to detect PRV and compare the changes of the relative content of PRV antigen before and after purification.It was determined that this method has the advantages of specificity,sensitivity and stability.The results of SDS-PAGE electrophoresis and Western blot showed that the protein expression level of purified PRV was higher than that of pre purified PRV,and the protein was also purified;The experimental results of double antibody sandwich ELISA showed that this method could accurately detect PRV,which could react with PRV but not with other pathogens;According to the results of double antibody sandwich ELISA,the OD value of purified PRV is greater and the reaction color is darker than that of pre purified PRV.It can be concluded that the antigen content of purified PRV is relatively increased than that of pre purified PRV.The purification method provides a method to increase and purify protein for the inactivated vaccine to prevent PRV,and provides a new idea for the inactivated vaccine to prevent pseudorabies.The establishment of double antibody sandwich ELISA provides a simpler,rapid and widely tested method for the accurate detection of PRV.