Establishment Of Indirect ELISA For Detection Fox Pseudorabies

Pseudorabies?PR?is an acute infectious disease caused by Pseudorabies virus?Pseudorabies virus,PRV?that endangers animal reproductive system and nervous system.Pseudorabies virus can infect a variety of mammals,such as pigs,rats,raccoon,mink and foxes.In 2011,there was an outbreak of PRV in pig farms in several provinces of China,and the epidemic of the new strain caused the Bartha vaccine strain is difficult to provide comprehensive protection of pigs.The new strain not only infringes the pig population,but also can infect the fur animals.Neurological itching,dyspnea,mental mania or depression and other clinical symptoms as well as the"regression fever"can be identified when the fox infected with PRV,all of which result in a high mortality of fox,which brings huge economic loss to fox breeding industry.In order to better prevent and control the disease,we developed an indirect ELISA test by virus isolation,prokaryotic expression of gB protein and preparation of HRP secondary antibody,which contribute to the detection of PRV from the fox.In this study,30 brain tissue samples from foxes suspected of PRV infection were collected during 2017²018.Six samples were confirmed positive by PCR test,with a 20%positive rate.Virus isolation was performed on the positive samples,and the isolated strain was verified by PCR test and indirect immunofluorescence assay,which was named HBfox-25.The virion morphology of HBfox-25 was observed by transmission electron microscopy after purification.TCID₅₀ of the virus was10^-5.6/0.1mL.In addition,we performed animal regression experiment and the fox appeared neurological symptoms,itchy skin and ripped the inoculation site,the foxes died 5 days after challenge test.In this study,the gB gene of HBfox-25 was amplified by PCR,and the target fragment was cloned into pET-32a vector,and the recombinant plasmid pET-32a-gb was obtained.Secondly,the recombinant plasmid was transformed into E.coli?BL21?,37?was used for shaking bacteria,and when the fungus was grown to Bacterial logarithmic phase,the expression was induced by IPTG.Finally,the purified protein concentration was 1 mg/mL as measured by an ultraviolet spectrophotometer.The optimal coating concentration of the antigen was determined by checkerboard titration.This study was a crude extract by saturated ammonium sulfate method fox serum IgG,was purified by column in turn.Finally,the purified rabbit IgG was immunized with Rabbit anti fox secondary antibody,and the purified two anti HRP was labeled by improved two step method.Finally,the indirect ELISA test for fox source PRV detection was established and the experimental results show that the indirect ELISA test established in this study has good specificity,sensitivity and stability.The establishment of this method provides new technical support for the diagnosis and epidemiological investigation of Fox PR.