Expression Of Pseudorabies Virus Glycoprotein E Epitopes In Picha Pastoris And Preliminary Application

Pseudorabies virus (PrV), is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. PrVinfection could cause its natural and main storing host--swine (also its maininfectious origination) a huge loses, the most huge economical lose in all swine infectious diseases except Classic Swine Fever and Food and Mouth Disease. 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, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system. This study aims to develop a better diagnostic antigen and method to distinguish g E positive and negative sera.Using a pair of primers designed according to the relevant nucleotide sequence from GenBank, the specific genes encoding PRV-sh gE epitopes were amplified. The PCR products of 660bp were cloned into P. pastoris secretory expression vectors p PICZ C.12 recombinant strains with specific genes encoding PRV-sh gE epitopes were obtained and when induced with methanol, the recombinant protein was successfully expressed in the supernatant, which is proved by SDS-PAGE. Western blot analysis showed that products had activity.The relations between expression level and growth condition were studied and the optimal expression conditions of the recombinant protein were: pH of BMMY medium 6.5, the cell density (OD600) 1.5, incubation for 72h, the concentration of methanol 1%. After induction under the optimal conditions, the concentration of the recombinant protein was 174. l g/ml. It was found that the supernatant should be conserved in -20 C after comparing of different temperatures on degradation of recombinant protein.After 10 passages, the recombinant strain still could express the target protein.The result indicated that the recombinant strain was stable in heredity.An indirect ELISA using the recombinant protein as coating antigen was carried out to distinguish the PRV gE positive and negative sera. The result showed the capacity of distinguishing gE positive and negative sera was very restricted, and the further study should be processed.