Fa Strain Of Pseudorabies Virus Ge Gene Expression In Escherichia Coli Of Ge-elisa, Differential Diagnosis, Methods Of Research

A 1.7Kb fragment encoding gE of PRV Fa strain was obtained by PCR from plasmid ppgE templated using a pair of the designed primers containing EcoRI and BamHI'sites. The gE gene fragment cutted with EcoRI and BamHI was inserted into the expression plasmid PBV220 including these two endonuclease sites for constructing the recombinant plasmid PBVgE. Strain DH5a of E. coli contain PBVgE was induced at 42 for 4-6hr after incubation with vigorous shaking at 30 for 3hr or so. A unique protein band was characterized in the lysate of the transformed bacteria by SDS-PAGE electrophoresis. The expression level was up to 17% of the total bacterical proteins. Western-blot analysis showed the expressed protein was specific to antisera against PRV Fa strain.The gE-ELISA for differentiation of PRV infected from vaccination was developed using the expressed gE protein as antigen. Before the serum samples were added. They were preincubated with the supermarant of the normal abstracts of E. coli for 30 min at room temperature. The gE-ELISA method was established by selecting the conditions with the coating concentration of gE antigen to be 6. 6ug/ml. the optimal dilution of serum to be 1:40. It revealed a negative reaction with positive sera of PRV HCV,PRRSV,JEV and SA215. It revealed a positive reaction with PRV standand positive serum. The results showed that the established gE-ELISA has the advantages shch as high sensibility strong specificity and good repeatability and can be used to differentiation of PRV infection from vaccination.