The Establishment,Characterization And Study The Role Of Eukaryotic Expression System Of B19 -XA VP1 Unique Region In Immune

Parvovirus B19, a small non-enveloped member of the genus erythrovirus,was discovered by Cossart et al.in 1975.Since the first discovery, studies have shown that the incidence of many diseases associated with B19 virus infection.B19 virus is wrapped by an icosahedral capsid with a single-chain DNA (5596bp) .B19 virus nucleic acid is a linear single-stranded DNA molecule which is a non-coated and there are many physical and chemical tolerance . The general method of virus inactivation can not prevent its spread. B19 virus gene encodes a major non-structural protein (NS1), two structural proteins (VP1 and VP2) and two small molecular weight proteins (7.5kDa and 11kDa) The only difference between VP1 (83kDa) and VP2 (58kDa) is the 227 amino acids in N terminal which is called VP1u protein. VP1u protein which has PLA₂ activity can hydrolyze fatty acids and phospholipids. VP1u protein appears to stimulate the immune disorder. Most VP1u virus capsid protein fragment extends to the outside and is the major antigen to stimulate the body to produce neutralizing antibodies. VP1u protein plays a key role in the pathogenesis of B19 virus infection.It may also become the preferred vaccine antigens of B19. The VP1u nucleotide of B19 XA virus existed variation. In view of VP1u protein's special role, B19 virus has its unique immunological characteristics. In 2009, our study confirms, animals immunized with VP1u protein not only can cause heart, liver, synovial tissue and other pathological changes, but also can lead to animals aspartate aminotransferase (AST), lactate de-Hydrogenase (LDH), creatine kinase (CK), MB isoenzyme of creatine kinase (CK-MB) and other enzymes increased. This may attribute toVP1u PLA₂ activity of the protein.It also may be the cross-reaction between the animal molecule and VP1u protein. However, it makes a question on using VP1u protein as B19 virus vaccine. Given the DNA vaccine and common protein vaccine, the protein which produced by DNA vaccine is more like the natural protein antigen. The vaccine can cause long-lasting host immune response.therefore, we design experiments in this item.Objective:Purpose of this study is to build a B19 virus VP1u eukaryotic expression vector.To identify recombinant vector by gene sequencing and the eukaryotic protein transfection.Then the recombinant vector is used to immunne animals. Cardiac tissue of immune mice for pathological examination; the number of CD4⁺ T cells and CD8⁺T cells is analysised by flow cytometry. ELISA (Enzyme linked immunosorbent assay, ELISA) test serum antibodies in mice immunized with the vaccine to determine the likelihood as a DNA vaccine.Methods:1. Construction of recombinant plasmid pIRES2-EGFP-VP1u. The VP1u gene of B19 virus was obtained from the prokaryotic expression vector.It was cloned into the eukaryotic expression vector pIRES2-EGFP.The recombinant plasmids were identified by gene sequencing and enzyme digestion.2. Recombinant plasmid pIRES2-EGFP-VP1u eukaryotic transfection and expression of protein. The recombinant plasmid were transfected into HeLa cells.Then Western blot and fluorescence microscopy were used to identify the expression of VP1u protein and green fluorescence protein.3. Recombinant plasmid pIRES2-EGFP-VP1u Immunology. Recombinant plasmid pIRES2-EGFP-VP1u was used to immunize BABL / c mice by intramuscular. In order to determine the feasibility as a vaccine, the number of CD4⁺ T cells and CD8 + T cells were counted by flow cytometry. Western blot and ELISA were used to detect antibodies in serum. Cardiac tissues of mice were detected by light microscope and electron microscopy.Results:1. The VP1u gene was cloned and pIRES2-EGFP-VP1u vector was build. The VP1u gene could express successfully in Hela cells. Double restriction enzyme digestion and gene sequencing proved the vector kept the same nucleotide in pQE30-VP1u prokaryotic expression vector in VP1u and did not produce the mutation.2. Immunofluorescence and Western blot ensured HeLa cells which was transfected by the recombinant plasmid could express VP1u protein.3. The number of CD4⁺T cells had a significant change in the second and third month.It gradually returned to normal in the fourth month after vaccination. But CD8 + T cell did not result in significant change. Cardiac tissues examination by light microscopic and electron microscopic did not suggest pathological changes. ELISA could detect specific antibodies against B19 virus VP1u.Conclusion:1. The recombinant plasmid contained parvovirus B19-XA VP1u gene was successfully constructed and expressed VP1u protein in HeLa cells.2. BALB / c mice could produce specific antibodies against B19 virus VP1u. Cardiac tissues by light microscopic and electron microscopic examination found no pathological changes. It suggested us that autoimmune disorders did not occur after the recombinant plasmid immunized animals.3. The number of spleen CD4⁺ T cells had a significant change in animals with DNA vaccine immunized. It suggested us that VP1u protein played an important role in humoral immunity.