Development Of An ELISA Method Differentiating Infected From Vaccinated Animals Of PRV

Porcine pseudorabies virus?PRV?mainly transmitted across the digestive tract,the respiratory tract,the air and the placenta.After infection,pigs may carry PRV for a lifetime and continuously drain them to outside.At present,there is no effective treatment for this kind of disease,which brings huge economic losses to the swine industry.At the end of 2011,there were suspected PR outbreaks in pig farms in Shandong,Henan and Hebei provinces that were immunized with certain PRV vaccines.The pigs developed high temperatures?40⁴2°C?,cough,diarrhea and systemic neurological symptoms.The newborn piglet mortality is high.The result of virus isolation showed that the pathogen causing this disease is a variant PRV,which raised new challenges for the prevention and control of pseudorabies?PR?.The PRV genome can encode a variety of proteins,gE protein is the main virulence factor,which plays a vital role in the transmission of the virus across the neuron.The advantage of choosing the gE protein as a target differentiating infected from vaccinated animals of PRV is that the gE gene is highly conserved,expressed in all field strains.And the antibodies of gE glycoprotein can be rapidly produced and lasted for a long time.So gE protein is commonly used as the first target for differentiating infected from vaccinated animals of PRV.Currently,gE gene is depleted in most commercialized PRV attenuated and inactivated vaccines.Because of the presence of field strain infected pigs,the gE gene depleted vaccines alone can not eradicate PRV properly.Therefore,combining with the differential diagnosis of infected from vaccinated pigs,eliminating PRV field strain infected pigs is the fundamental measure in the process of PRV purification.At present,China relies mainly on foreign expensive kits to differentiating infected from vaccinated animals of PRV.Although some domestic companies also have similar kits for PRV detection,there is a certain gap for the effects compared to foreign imports.Therefore,it is necessary to develop a domestic kit for the accurate identification of differentiating infected from vaccinated animals of PRV.In order to establish an indirect ELISA method for PRV antibody detection,this study amplified the PRV gE gene dominant epitope fragment gE_37²43 by PCR and connected it with pET-32a?+?vector to construct recombinant plasmid.After the optimization of protein expression condition,the soluble expressed gE_37²43 protein was obtained.The mice were immunized with gE_37²43 protein by Ni-NTA affinity chromatography purified,two kinds of monoclonal antibodies of PRV were obtained after purification with ammonium octanoate,so as to lay a foundation for the development of PRV monoclonal antibody blocking ELISA.An indirect ELISA method for detection antibodies of PRV was established using the purified gE_37²43 protein as a coating antigen,and optimizing the condition of antigen coating process,serum diluting,sealing liquid,the work concentration of antibodies,reaction time,the ELISA method established in this study has a sensitivity of 93.33%and a specificity of 93.75%,the intra-assay and inter-assay coefficients of variation are less than 10%,the coincidence rate with the commercial kits is 92.00%.Therefore,compared with commercial kits,the PRV antibody detection indirect ELISA method established in this study also has a great sensitivity,specificity and detection rate,which can provide a technical support for domestic differentiation infected from vaccinated animals of PRV.