Cloning, Sequence Analysis Of Unknown Regions Of Pseudorabies Virus And Expression Of Related Genes

Pseudorabies virus (PRV) is the causative agent of Pseudorabies (Aujeszky's disease), a serious infectious disease of several domestic and wild animals. The natural host and reservoir for this virus is the pig. Since Pseudorabies was first reported in 1902, it has spread throughout the world and has become one of the most economically important diseases of the pork-producing industry. Like other alpha herpesvirus such as herpes simplex virus type I, varicella-zoster virus and bovine herpesvirus type I, PRV also has some typical characteristics of alpha herpesvirus, including latency and neurotropism. PRV is more advantageous for the research of the production and distribution, neurotropism and pathogenesis of alpha herpesvirus and evaluation of immune response and new vaccine. Therefore, PRV has interested the researchers and become an excellent model system for herpesvirus biology.Although cloning and sequencing of some important functional genes of PRV were carried out in the middle of 1980s, sequencing of PRV genome has developed slowly because of high content of G+C of PRV genome (the mean content of G+C is 74%). In recent years, genomic research of PRV has been strengthened with the progress of sequencing technology and the requirement of the investigation of functional genomics. Compared with HSV-1 genome, the combined sequence from various PRV strains published before 2001 includes 95% of the entire genomic sequence of PRV, and only the sequences of three gaps remain unclear, including UL48-UL49, UL15-UL17 and UL31-UL37. Cloning and sequencing of genes of these gaps are very significant for researches of the structure and function of PRV genome.In this study, two gaps of PRV Ea genome (UL48-UL49, UL15-UL17) were cloned and sequenced, and some genes of the gaps were expressed. The following researches were explored.Sail 2.8kb fragment of PRV Ea strain containing UL49 gene was obtained by southern blot using UL49.5 gene as probe. Sequencing of Sall-BamHl 2.0kb fragment in this region indicated the 2.0kb fragment is 1959bp long, containing UL50 (partial), UL49.5, putative UL49 and UL48 (partial) genes.UL48 gene was cloned by PCR using primers designed according to sequence of partial UL48 and 5' end of PRV Ka UL47. Result of sequencing showed that UL48 contains 1242bp and encodes 413 amino acids, the middle of which is homologous to VP16 of other Alpha Herpesvirus. Sequence of the gap of UL48-UL49 are made clearentirely based on sequencing of UL48 and SaR-BamHl 2.0kb.Prokaryotic expressing plasmid pGEX-KG was constructed by inserting UL48 into prokaryotic expressing vector pGEX-KG. GST-UL48 fusion protein was expressed efficiently in E.coli with apparent MW of 65kD. Eukanyotic expressing plasmid of UL48 pcDNA-UL48 was constructed and transfected into Hela. In Western blot experiment, several bands of Hela expressing UL48 with apparent MW of 38kD and 50-60kD could be seen, which proved UL48 had been expressed in Hela and may interact with components of the cell. In order to study localization of UL48, Eukanyotic expressing plasmid pEGFP-UL48 expressing EGFP-UL48 fusion protein was constructed. Fluorescence of Hela cells transfected with pEGFP-UL48 indicated that intracellular localization pattern of UL48 was variable.UL49 gene was cloned by PCR, inserted into prokaryotic expressing vector pET-28a, and then expressed efficiently in E.coli with apparent MW of 37kD. Eukanyotic expressing plasmid pcDNA-UL49-M4 expressing UL49-mut4EGFP fusion protein was constructed and transfected into Hela. Detection of direct fluorescence revealed that the localization pattern of UL49 was multiple too, but UL49 localized predominantly in the nucleus or peri-nucleus with dotted pattern.In order to determine the sequence of the gap of UL15-UL17, PRV BamHl-3 and BamHI-4 fragment were cloned using restriction method and sequenced segmentally. Sequence analysis of UL13-UL17 indicated that UL16 and UL17 genes of PRV are inside UL15, probably composing intron of UL15. Primers of partial UL15 was designed according to the pu...