| Pseudorabies virus (PRV) is a member of the genus of Varicellovirus of the family Herpseviridae, subfamily Alphaherpesvirinae, which can cause actue infection disease to many domestic and wild animals. Pigs are the natural host and the source of infection to it. After infecting PRV, pigs have serious diseases with nervous and respiratory system, futher, with reproductive failure. PR has inflicted major economic loss in pig industries worldwide,so it is important to prevent and control and eradicate it.But PRV has latent infection in pigs which carrys huge difficulty to eradicate it.In the part one of this study,the early protein 0 (EP0) gene which is rated with the latent infection was cloned,sequenced and expressed.PRV EPO gene is an early protein, which is located at the end of the unique long region of PRV genome and contains the RING finger domain in the ammo-terminal region with homology to ICP of HSV-1, which can activate transcription form the PRV TK, gG gene promoters and is nonessential for PRV replication but may be important for reactivation from the latent state. In this study, the 1.23kb DNA fragment encoding the early protein EPO of pseudorabies virus Ea strain was amplified by PCR technique and cloned into pBluescript II sk+. Three sequencing plasmids which containing the partial fragment of the EPO gene were constructed and the sequence were obtained by Sanger's dideoxgen termination method. When compared with. PRV InFh strain, there were multiple site-mutations a deleted-mutation in the EPO gene of PRV Ea strain, and the diversity of arnino acid residues also existed. Then, the EPO gene was inserted into prokaryotic expression vector, PET-28a, fused into the downstream of 6XHis-Tag in frame,to yield the expression plasmid pETEPO. After induction by IPTG, a high expression of fusion protein was obtained, SDS-PAGE analysis and Western blotting showed that the fusion protein was 62KD and the protein was specific to antisera against PRV Ea strain. This indicated that the EPO gene be expressed in BL21(DE3) and the expression products have immunogenicity. The EPO gene was further cloned into BamHI and EcoRI sites of eukaryotic expression vectorpcDNA3.1+ and resulted to a eukaryotic expreesion plasmid pcDNA-EPO. The expreesion plasmid transfected into the IBRS-2 cell and the cell lines expressed stablely EPO was established by the pressure of G418. Expression protein distributed on the membrane and had biology avtivity by the diagnosis of ELISA.Pseudorabies virus (PRV) and Porcine parvovirus (PPV) are the major pathogenys to cause reproductive failure, they often infect swine together, so it is necessity to prepare the recombination vaccine of PRV and PPV.The genome of PRV is a linear DNA molecule of about 150kb,which includes many nonessential genes into which foreign genes can be stably inserted. This makes attenuated PRV become a promising candidate for the development of a live vaccine vector. In the part tow of this study , the primers for PCR with PPV VPa gene were amplified by PCR technique and cloned into the BamHI site with the downstream of gG promoter which was contained in plasmid pUSK,which was the basement to construct the PRV recombinants that expressed VP2 of PPV.
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