| 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 program. 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 diagnostic method for the pseudorabies eradication campaign in China and provide specific monoclonal antibodies against PrV to study structure-function and molecular pathogenesis of PrV, the following research were explored.1. The expression of PrV gE gene in Pichia pastoris.A 717 base pairs long artificial gene fragment of PrV gE has been synthesized by using preferred codons in the highly expressed Pichia genes. Four groups of oligonucleotides were successively cloned in pMD18-T vector to obtain four large fragments. Ligation of these four fragments resulted in a cloned DNA MgE717 coding for the 29-268 amino acid region of gE. Sequencing result showed that there is a mutation at 255 amino acid, Asn instead of Asp.The modified gE fragment MgE717 was then inserted into pPICZ a A vector, fused with the a -MF factor signal sequence and the c-myc epitope 6 His to construct an expression plasmid pZ a AMgE717. After transforming pZ a AMgE717 into P. pastoris cell SMD1168H, one recombinant named SAMgE717-6 was obtained for highest expression yield. The expressed protein of SAMgE717-6 was not stable and started to hydrolysis three days later. Glycosylation analysis revealed that the expressed product has been assembled with N-linked oligosaccharides.The truncated gE gene encoding 39-268 amino acid region of gE was amplified by PCR from pSDM1.78K+ plasmid harboring PrV Ea strain gE full length DNA. The gE fragment was then inserted into pPIC9K vector and fused with the a -MF factor signal sequence to construct an expression plasmid p9KgE687. After transforming p9KgE687 into P. pastoris GS115 cell, the transformants were screened by G418 for multi-copy recombinants. A multi-copy recombinant was obtained, named as G17. Western-blot analysis showed that the expression product of G17 was 35 kD. After processed by PNGase F, no change of molecularweight was observed.Expression vector pZ a BgE687 composed of the original gE sequence and pZ a AMgE687 composed of the modified gE sequence were constructed respectively. Both the foreign fragments in two vectors coded 39-268 amino acid region of gE. After transforming the two expression vectors into SMD1168H, the recombinants were screened by ELISA. SBgE687-7 and SAMgE687-4 were obtained respectively for the highest antigenic quantity. But no expressed product was observed in induced supernatant of SBgE687-7 by SDS-PAGE and Western-blot analysis. In supernatant of SAMgE687-4, a specific expressed product was observed by Western-blot analysis. N-linked glycosylation analysis showed that N-linked glycosylation was assembled on the expressed product. This demonstrated the recombinant protein product was improved by using the bias codon of highly expression protein in P. pastoris.2. Development a gE-ELISA for gE-specifical antibodies differential detection.A gE-ELISA was developed using expressed gE in P. pastoris. The suitable concentration of antigen and the serum dilutions was selected by titration test. The cut-off value was determined by testing 64 negative sera. The gE-ELISA is of good stability and specificity. Comparison between gE-ELISA and imported PrV gpl antibodies diagnosis kit showed the two methods had 90.8 percent agreement, implied that gE-ELISA is an effective method for detecting...
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