Cloning And Expression Of GK Gene, UL54 Gene, ORF-1 Gene Of PrV Ea Strain BamHI5' Fragment

Pseudorabies Virus, the causative agent of PR of many domestic and wild animals,belonged to the alphaherpesvirus subfamily of the herpesviridae, which caused serve financial losses in the world pig industry. In order to get deeper understanding of the melocular pathogenic mechanism of PrV and finish the PrV genome plan eariler, we selected the BamHIS' fragment which is located on the left of PrV genome and was quite different between a-herpesvirus, cloned, sequences analysis and expressed three whole genes gK, UL54, ORF-1 which BamHIS' contained , based on the PrV Ea strain which was isolated and identificated from Hubei provine by animal virology lab of Huazhong agriculture university.Glycoproteins involved in the replication of PrV, determinded PrV s virulence, induced the protect and immunity. Now, there were 11 glycoproteins of PrV had been identified. Glycoprotein K was the component of PrV viron, and was essential for replication, involved in egress of PrV particles. During the replication of PrV, it had only one immediate-early gene, IE180, and four early genes. The early genes involved in regulatory of expression of early genes and late genes. Among those early genes, UL54 was an important early regulatory gene, homologous to ICP27 of HSV-1. PrV UL54 regulated the expression of leaky late and late genes on transcription and post-ranscription leve, and maybe it was related to apoptosis of PrV. The ORF-1 gene was distincted between PrV and EHV-1 Ab4. Neither gene was essentional for replication. The function of ORF-1 was not very clear, maybe it was related to virulence of PrV.Three pairs of primers were synthesized according to the sequenceof PrV Ka strain. The fragment about 939bps, 1086bps, 624bps,containing gK, UL54, ORF-1 genes encoding domains were ampified by polymerase chain reaction(PCR), using BamHI5' fragment of PrV Ea strain as template. The three genes were cloned into pBluescriptIISK+, resulting recombination plasmids pSKgK, pSKUL54, pSKORF-1.The sequences were obtained by Sanger' s sequencing technique and compared with PrV Ka strain using blast software. The homologs of gK, UL54, ORF-1 between Ea strain and Ka strain were 97. 78%, 97. 24%, 94. 88%. The predicted secondary structure showed that gK was a high hydrophbic glycoprotein,containing a singal peptide, four transmembrane domains and two potential N-glycolated site. The early protein, ICP27, which was encoded by LJL54 gene has a conserved classic zinic finger, which was the binding site of DNA, RNA and protein. The ORF-1 gene only esisted among PrV and EHV-1 Ab4 strain, that the homolog of the two strain was 35%.The whole gK,UL54, ORF-1 genes were cloned into porkaryote expression vector pGEX~2T. After cut two hydrophic domains of C-terminal, the partial gK gene containing 588bps was cloned to pGEX-KG. After induction by IPTG, the whole gK, UL54, ORF-1 genes didn' t express while the partial gK gene expressed a 40 KD fusion protein at a high level.The gK, UL54, ORF-1 genes were cloned into eukaryotic expression vector pEGFP-Cl, following the EGFP(Enhanced Green Fluorescent Protein). The pEGFP-Cl and recombinant plasmids were transfected into BHK-21 cell with lipofectin and selected under G418 , until the normal BHK-21 cell die out. Expression protein were detected under inverted fluorescence microscope. The results show that all of the gK, UL54, ORF-1 genes were expressed on BHK-21 cell. The gK and ORF-1 genes were expressed in the cytomembrane and cytoplasm of BHK-21 cell while UL54 gene was expressed in the nuclear of BHK-21 cell.