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According to the sequence of gL gene of Pseudorabies Virus Strain Ea published in GenBank, (GeneBank accession No. AF448456), a pair of specific primers F1 and F2 were designed by oligo6.0 and premier 5.0 program. The genome of Pseudorabies Virus Strain SL was used as template to amplify gL gene. Then the amplified products were subcloned into pMD-19-T vector, and the recombinant plasmid was designated pMD-gL. It was confirmed positive by restriction enzyme analysis and sequenced by Beijing Genomics Institute. The open reading frame of gL gene consisted of 471 base pairs(bp) capable of 149 amino acid residues and that of different isolates were conserved highly, but within herpesvirus, wild SL strain shared the most 14.2% homology with EHV-1, therefore it was indicated that the herpesvirus was less conservative. To a significant extent, the analysis of gL gene also showed codon usage bias. Besides, there was an obvious CpG island between 48 ~ 414bp. Comprehensive analysis of gL protein, molecular formula of gL protein was C757H1167N193O217S4, and there was a signal peptide of 16 amino acid residues, 4aa~ 22aa, 101aa~ 123aa for the transmembrane. Protein subcellular localization manifested 55.6% of the gL protein in the membrane and also distributed in the follicular and mitochondria. There existed about 3 hydrophilicity and hydrophobicity peak of gL protein , and mature peptide hydrophobic sequence reflected in the 88-118 amino acids site in particular. Comprehensive analysis of epitope revealed that gL protein epitopes were located in the 27aa, 36aa ~ 37aa, 42aa ~ 43aa, 44aa ~ 47aa, 66aa~ 69aa, 80aa ~ 81aa, 96aa, 142aa ~ 143aa, 149aa ~ 154aa. The characterization of gL of PrV SL strain provide the basis for additional comparative studies on alphaherpesvirus glycoprotein function. And the study of gL protein should help to shed more light on the importance of both proteins in a natural herpesvirus-host system.