Expression Of The Main Antigen Region Of GE Of PrV And The Study On The Diagnosis Method Of Indired ELISA With The Expressed Protein

Pseudorabies virus(PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease),one of the most serious infectious diseases of several domestic and wild animals. Swine was the natural host and reservoir of PrV. Pseudorabies was an economically important disease of swine industry. Currently it is an important task to prevent, control and eradicate this disease in China. Use of a combination of an effective gE gene-deleted PrV vaccine with a companion diagnostic kit for PrV glycoprotein gE has proven successful in several Pseudorabies-eradication programs. Meanwhile, although gE was unessential for viral replication in cell culture, it played an important role in determining virulence and neurotropism.In order to develope a PrV gE differential diagnostic method for the pseudorabies eradication program in China, the following researches were explored.1. The expression of the major epitope domain of glycoprotein gE of pseudorabies virus in E.coli.A 0.63kb DNA fragment encoding the major epitope domain of glycoprotein gE of pseudorabies virus MinA strain was inserted into the downstream of the T7 promoter of an expression vector, pET-28a, to yield the recombinant plasmid pETgE630. After induced by IPTG, a high level expression of fusion protein was obtained. SDS-PAGE and Western-blotting analysis showed that the fusion protein was 29kD and the protein was specific to antisera against PrV MinA strain.2. purification and renaturation of expression proteinThe inclusion bodies in E.coli with plasmid pET-28a , which have the higher expression level, were obtained by sonication of the bacterial cells, from which the purified activative expressed recombinant gE630 proteins were got by urea and TritonX-100 washing and by renaturing, refolding and dialyzing. The results of SDS-PAGE show that washing by urea and Triton X-100 can decrease the amounts of bacteria proteins in theinclusion body. The dissolving effect of urea for the inclusion body is good, which eventually resulted in 85% of purity of recombinant gE630 protein. 3.The development of gE-ELISAThe gE enzyme linked immunosorbent assay (ELISA) based on the recombinant glycoprotein E which have been purled, denatured, and renatured...