Genetic Characters Of A Prrsv Isolate With Deletion In GP5and The Construction Of The Baculovirus Vector Of GPS And M Protein

Porcine reproductive and respiratory syndrome (PRRS), caused by Porcine reproductive and respiratory syndrome virus (PRRSV), is considered to be one of the most important infectious diseases of swine that has been devastating the swine industry since the late1980s. In China, the first case of PRRS was reported in1996and the disease was encountered in almost all provinces. Since then,PRRS has become one of the most significant problems for swine industry. In2006, the high-fever syndrome caused by the highly pathogenic PRRSV (HP-PRRSV) broke out in the south of China, resulting in great economic losses to the swine industry. The main goal of this study is to raise a epidemiological investigation of "swine highly fever disease" in Shaanxi and provide a basic research for the research and development of new PRRSV vaccine. In this process, we isolated a PRRSV with deletion in GP5from Hanzhong of Shannxi province, in order to research the genetic variation and molecular characteristics of the virus, we analysed the GP5and NSP2genes. According to the present situation of the high variability of PRRSV that has limited the effect of vaccines, we designed a recombinant baculovirus vector for the new vaccines to ease that problem and laid the foundation for subsequent research.The main research are as follows:1In order to understand the variation of porcine reproductive and respiratory syndrome virus(PRRSV) in Shaanxi province, the virus was detected from swine clinical samples, NSP2and ORF5genes were amplified, cloned and sequenced with RT-PCR for analyzing the genetic variation of PRRSV.The result shows that the strain belongs to the North American genotype, named HZ1007. It has discontinuous30amino acids deletion in the aa position of481and533～561of NSP2, which is the same deletions with the highly pathogenic PRRSV. Moreover, the ORF5gene of HZ1007strain contains a continuous11amino acids deletion in the aa positon of85～95.PRRSV HZ1007is highly homologous to HUN4and SY0608strains. Phylogenetic tree analysis reveals that HZ1007has closer relationship with the highly pathogenic PRRSV than classical strains, its may be a new variant of the highly pathogenic strain. The pathogenicity and biological characteristics need further research and analysis.2In this study, depending on the advantages of the new baculovirus expression system(MultiBac system), target genes could be surface displayed and expressed at the same time by the recombinant virus. In order to improve the penetration targeted and antigenicity of the virus, there are two components which has been inserted in the vector to be displayed, Flagellin and InvasinC. A potent universal helper T-lymphocype epitope has been spliced between the neutralizing and nonneutralizing epitopes in the N-terminus of GP5gene. And we also removed the glycosylation sites of GP5. The plasmid was identified by the method of enzyme digestion and PCR. In the meantime, we designed a control plasmid, in which the functional components were replaced by the fluorescent proteins. With the control plasmid,we packaged the virus and validated the function of recombinant virus.The PRRSV with deletion GP5was first discovered in this study, that provided an important reference for the research of PRRSV genetic evolution and immunization. Meanwhile, according to the high variation extent of the GP5and the protective capacity of PRRSV vaccines is not enough, we designed a vector for a novel baculovirus vaccine.