Expression Of Major Epitope Domain Of Pseudorabies Virus Glycoprotein E In Escherichia Coli And Application Of The Expression Product As Antigen To Develop An Indirect ELISA For The Detection Of Viral Glycoprotein E Antibodies

Pseudorabies virus (PRV), is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. many countries in Europe and America have carried out"eradicating project", and Glycoprotein E of Pseudorabies virus is known to be an important diagnostic antigen in Pseudorabies eradication campaign.In order to obtain the gE antigen in large quantity and at low cost, the gene encoding the major epitope domain of gE was expressed in Escherichia coli expression system, then the gE-ELISA for differentiation of PRV infected from vaccination was developed by using the recombinant gE protein as antigen.The gene encoding the major epitope domain of glycoprotein E of Pseudorabies virus was amplified by PCR and cloned into PMD18-T vector, then subcloned into the downstream of T7 promoter of an expression plasmid, PET-32a. After induction by IPTG, the fusion protein was expressed in Escherichia coli BL21. SDS-PAGE and Western blot analysis showed that the purified fusion protein measured 38ku could react with pig serum containing antibody against PRV. All results indicate that the recombinant fusion protein can be used as antigen of diagnostic assay to detect antibody against PRV.The gE-ELISA was established by using the recombinant protein as antigen, and all reaction conditions of this gE-ELISA were explored, the optimal coating concentration was 1.7ug/ml, the optimal coating condition was at 4℃over night, the serum sample was diluted to 1:40, serum sample and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃for 1 hour, the substrate was added and incubated at room temperature for 10 min before terminated with stopping solution.172 swine serum samples were detected for the gE antibody by using this developed indirect gE-ELISA and the INGENASA gE Antibody Test Kit. It was found that the specificity and sensitivity of the developed indirect gE-ELISA were 93.4% and 93.8% respectively, the correlation between these two ELISA was 93.6%. In addition,...