Study On Virus Vector Vaccine And DNA Vaccine Coexpressing E And M Protein Of Porcine Reproductive And Respiratory Syndrome Virus Fused With VP22 Of Bovine Herpesvirus 1

Porcine reproductive and respiratory syndrome (PRRS) , caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the United States in 1987, PRRS has already been one of the most economically important diseases of the global swine industry. Vaccine inoculation is the main method to prevent and control this disease. Currently, the commercial vaccines to prevent PRRS are mainly attenuated and inactivated vaccines. Although attenuated vaccines can provide protection against PRRSV, there exist dangers of reversion to a more virulent strain. Compared with attenuated vaccine, inactived vaccine is safe, but its effect is not stable. Therefore, it is urgent to develop safer and more effective and cheap novel vaccines to prevent and control PRRS.Protein transduction, a unique biology phenomena, was discovered in recent years. Proteins which have protein transduction property can penetrate the cell membrane and can be transported from the original expressing cell to surrounding nonexpressing cells. Acted as a new and vital technology, protein transduction offers a new idea and an efficient method for improvement of novel vaccines. Heretofore, it has been demonstrated that the VP22 homologues from alphaherpesvirus, e.g. herpes simplex virus type 1 (HSV-1), bovine herpesvirus 1 (BHV-1), etc., have the remarkable protein transduction property and have been used as immunoadjuvant to enhance the immunogenicity of DNA vaccines or virus vector vaccines, and BHV-1 VP22 could be considered as the most efficient immunoadjuvant.Based on the above research background, the two main immunogenicity genes of PRRSV, the ORF5M gene (the modified ORF5 gene having better immuogenicity compared with ORF5 gene) and ORF6 gene, were served as target genes, and BHV-1 VP22 as the immunoadjuvant, pseudorabies virus(PRV)-based vector vaccines and DNA vaccines of PRRS were explored in the present study. The main contents were as follows: 1. Construction of recombinant PRV TK~-/gE~-/VP22E~+/VP22M~+ coexpressing PRRSV E and M protein fused with BHV-1 VP22 and its immunoreactionThe complete coding regions of PRRSV ORF5M gene and BHV-1 VP22 gene having no stop code were amplified by PCR from the plasmids containing ORF5M gene or VP22 gene, respectively. And then ORF5M gene and VP22 gene were cloned into pMD18-T vector to generate the recombinant plasmids pMD18-ORF5M and pMD18-VP22. There was not any mutation on ORF5M gene or VP22 gene by sequencing. Both ORF5M gene and VP22 gene were inserted into a PRV universal transfer vector...