Isolation And Identication Of PRV LA Strain And Construction Of TK～- Recombinant PRV Expressing EGFP And CSFV-E2

1. The author isolated and identified one strain of pseudorabies virus (PRV) from a outbreak in a farm in Shandong Province and designated it as PRV LA strain, The isolate was characterized comprehensively. The virus was continually inoculated 5 generations onto rabbit kidney cell and chicken embryonic fibroblast cell (CEF) and induced typical cytopathic effect (CPE).The typical CPE cloud be seen when culturing the virus on RK-13 cell .PK-15cell .BHK-21cell and 143TK~ Cell and the titerof the isolated virus was 10652 TCID50/0.1mL and the typical pseudorabies virus particles could be found under electromicroscope. The isolate was sensitive to chloroform and ethylether and inactivated for 30 minutes at 56癈and could be neuralized by the positive serum to pseudorabies virus. The typical clinical syndrome could be observed when four rabbits and to Balb/c mice and three piglets were infected with the isolate. The sequence of 262 base pairs lying gD gene could be amplified with the DNA extracted from the isolate and a set of oligonucleotide primers. All of the evidences indicated that the isolated virus was pseudorabies virus.2. The gD gene of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pGEM-T easy vector and the nucleotides of the gD gene was sequenced. An analysis -of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1453 base pairs and it has one ORF of 1203 base pairs to have the potential to encode a protein of 400 amino acids. The gD gene of PRV LA strain shares highly conserved nucleotide with PRV Ea strain, Hubei strain and NIA-3 strain and Kaplan strain. The homology of deduced amino acids of PRV LA strain with PRV Ea strain. Hubei strain and NIA-3 strain and Kaplan strain is 97.8%, 97.8%,97.5%,98.1%,98.6%, respectively. But the PRV gD genes shared limited homology to other alphaherpesviruses equivalents: BHV-5.EHV-1 ,FHV-l,CaHV-l,MDV.HVT,ILTV,HSV-l,HSV-2 and SHBV.The nucleotide sequence analysis of the 6 PRV strains revealed that there was a highly repeated and altered region which lay to 802nt-837nt in their nucleotide sequencescorresponding to their amino acid sequences 267-279,the insertion and deletion of nucleotides in the region made the gD gene variable between 1197nt-1215nt.The homology analysis of deduced amino acids of PRV LA strain with other alphaherpesviruses equivalents showed that gD could separate into 4 groups: simplex viruses and varicella viruses and Marek's diseas-like viruses and infectious laryngotracheitis virus and the phlyogenetic tree analysis of the gD sequences may contribute futher evidence of the evolution of gD of alphaherpesviruses from a common ILTV gD progenitor.3. The thymiding kinase(TK) gene in the unique long region of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pUC 19 plasmid vector and the nucleotides of the TK gene was sequenced. An analysis of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1048 base pairs and it has one ORF of 963 base pairs to have the potential to encode a protein of 320 amino acids. The TK gene of PRV LA strain shares highly conserved nucleotide with PRV NIA-3 strain and Ea strain and SH strain. The homology of the nucleotide sequence of PRV LA strain with PRV NIA-3 strain, Ea strain, SH strain, HSV-1, VZV is 98.9%, 99.5%, 99.3%, 36.4% and 39.1%. respectively; and the homology of deduced amino acids among them is 98.4%, 99.7%, 98.7%, 36.6% and 37.2%, respectively. The amino acids of PRV TK contain the conserved ATP-binding motifs (-GXXGXGKT-) and the conserved nucleosite-binding motifs(-DRH-). The similarity of the two conserved bindings between PRV-LA TK and homo sapiens thymidylate kinase and mus musculus thymidylate k...