Construction And Identification Of PRRSV GP5 Protein Gene Vaccine And Preparation Of Three Molecular Adjuvant

Porcine reproductive and respiratory syndrome(PRRS) is capable of causing reproductive failure in pregnant sows and severe respiratory symptoms of an infectious disease, the disease pathogen is porcine reproductive and respiratory syndrome virus(PRRSV)the main target cells are porcine alveolar macrophages. The main route of transmission is airborne, and contact with infected semen spread of the virus is highly contagious, often causing serious economic losses to the pig industry. Glycosylated envelope protein(GP5)containing 2-5 glycosylation sites, is the major protective antigen of the virus and can neutralize the virus, the virus loses infectivity and is capable of inducing neutralizing antibodies, is novel DNA vaccine designed primarily target protein.Gene vaccine refers to the DNA vaccine, is about to exogenous antigen gene connected to a eukaryotic expression vector to construct a recombinant plasmid vector and then introduced into a host body, and expressed antigenic protein in host cells, stimulate the immune system to produce an immune response reaction. Within a certain time limit,continued expression of antigen genes in a host cell, activation of the immune response, to achieve the purpose of disease prevention. Inactivated vaccine, DNA vaccine compared to attenuated vaccine has a low production cost, easy storage and transportation, continued strong, safe, etc., for the disease has a good effect, it can induce a more effective immune response, but accounting vaccine antibody production is slow, low level of problem that requires effective adjuvant assisted to overcome these problems.Second messenger c-di-GMP, c-di-AMP, 2'-3'c GAMP as a signal molecule in bacteria found to play crucial in virulence of bacteria, metabolic balance of the cell wall, bacterial growth and biofilm formation an important role in eukaryotic cells, can be used as a signal molecule is recognized by the host, activation of the innate immune response. Second messenger function as an immunomodulator, very good to regulate the body's immune function, therefore, may have become immune adjuvant potential.The construct the expression of PRRSV GP5 gene regulation in eukaryotic expression vector and expressed in eukaryotic cells to do their research and experiment explored the c-di-GMP, c-di-AMP, 2'-3'c GAMP preparation and purification methods, specific contents are as follows:1. Construction and Expression of PRRSV GP5 Gene Regulation VerificationEukaryotic Expression PlasmidTotal RNA from laboratory to save porcine reproductive and respiratory syndrome virus,RT-PCR amplification to GP5 gene c DNA, cloned into the expression vector gene regulation p XJ41, transformed into E. coli Trans T1 competent, after sequencing enzyme digestion no mutation give p XJ41-GP5 recombinant plasmids were transfected into Hela cells by indirect immunofluorescence and Western Blot experiments show, p XJ41-GP5 recombinant plasmid capable of expressing in eukaryotic cells.2. The Preparation and Purification Methods of c-di-GMP, c-di-AMP, 2'-3'cGAMPVibrio cholerae picked VCA0956( the enzyme necessary for the synthesis of c-di-GMP),VC0179( the enzyme necessary for the synthesis of c-di-AMP)and containing mc GAS( the enzyme necessary for the synthesis of 2'-3'c GAMP) gene expression E. coli strain sequence,using Ni-NTA affinity chromatography prepared VCA0956, VC0179, mc GAS three protease,after SDS-PAGE was determined to verify protein protein; then the protease by molecular sieve chromatography three. In the case of the presence of the enzyme and substrate,according to c-di-GMP, c-di-AMP, 2'-3'c GAMP reaction formula, formulated reaction system to prepare three adjuvants, after exploring HPLC conditions to determine the rank and file agent peak time and purity, purification of the desired protein.The study of c-di-GMP, c-di-AMP, 2'-3'cGAMP and pXJ41-GP5 combined immunodeficiency enhancement PRRSV GP5 DNA vaccine basis.