Construction Of Recombinant Pseudorabies Virus Expressing The Rabies Virus Glycoprotein And The Transient Expression

Rabies is an important zoonosis that causes severe infection to the central nervous system and is usually fatal. In recent years, the serious problem has ascended tendency year by year in China.The reasons are all round. The main reasons are single type of vaccine and the low level of immunization. Lower vaccination coverage of dogs and stray dogs is widespread, largely because of poor awareness of precaution in rabies and the high cost of vaccination. So the control and eradication of human rabies depends on the development of safe, effective and economical vaccine that might be used in pre-exposure vaccination programs for animals.Long time ago, Genetically engineered rabies vaccine has been researched in our Lab.It is one example of the most successful genetically engineering vaccine. During prophasing reseach,we inserted the expression cassette of glycoprotein respectively, consisting of the human cytomegalovirus(CMV) immediate promoter, rabies virus strain 8202, SRV9, CVS glycoprotein cDNA and polyadenylation signal of the SV40. Then, we successfully gain three strain recombinant CAV-2 (CAV-2-8G, CAV-2-SRG, CAV-2-CVSG) through transfecting DK cells by Lipofectamine?.The result of immunity test indicated that the immunogenicity effectiveness of rabies was showed effective, but it was still lower compared with immunization with live or inactive rabies virus.The vector of recombinant vaccine is always adenovirus in previous researching.Because the adenovirus vector is limited capacity of exogenous gene and intensity respond in immune system that it can restrain or weaken the immune response to exogenous gene. Therefore, it is necessary that we research a suitable viral vector. PRV has the largest capacity in capacity of exogenome. Not only does it not infect people, but also does it latent infection cell.So PRV is one of the researching viral vectors.In previous, it contains only one expressing cassette in viral vector. but the neutralizing antibody level is not as high as that induced by the conventional vaccines, i.e., inactivated or attenuated live vaccines. It is broken though that viral vector contains only one expressing cassette , and it is constructed expressing cassette side by side in viral vector. In this way, not only have the theory materials been supply that researching capacity of PRV viral vector, but also has the level of CAV glycoprotein antibody been study that recombinant virus induces organism.Firstly, the glycoprotein of rabies virus strain 8202 was amplified by reverse transcription-polymerase chain reaction and cloned it into eukaryotic expression vector pIEGFP-CG, and obtaining the expression vector pI-dG. in my study. Meanwhile,the middle transfer vector p8AA has been reconstructed,and obtain the vector of p8-L. The resultant plasmid p8-dG has been gained that the eukaryotic expression frame of plasmid pI-dG does inserted plasmid p8-L. So the glycoprotein of rabies virus strain SRV9 does inserted eukaryotic exprssion vector pIRES1neo and obtaining the plasmid pI-SR.The eukaryotic expression cassette of the glycoprotein of rabies virus strain SRV9 cloned into vector p8-L and obtaining the plasmid p8-SR. The resultant plasmid p8-dG-SR has been gained and the eukaryotic expression frame of plasmid pI-dG does inserted plasmid p8-SR. Then the transfer plasmid p8-dG,p8-dG-SR were used to cotransfect the PK-15 cells respectively with lipofection transfection procedure .After six hours, The purified genomic DNA of TK-/gI- deleted strain PRV does added.But the EGFP gene was not expressing after six cycles of plague purification .In the experiment,we have reconstructed the plasmid p8-L,cloned the glycoprotein of rabies virus strain 8202 and constructed the transfer vector of p8-dG,p8-dG-SR. It bases advantageously to study genetically engineering vaccine and the capacity of foreign gene in Protein Kinase (PK) gene.We can't obtain recombinant virus.(But we have get the recombinant virus that partaked EGFP gene.). Thus we must raise the efficiency of the homologous recombination to PRV.