The Initial Research On The Structure And Immunizing Character Of Human Parvovirus B19-XA Unique Protein

Human parvovirus B19(B19),a member of Parvoviridae family and Parvovirinae subfamily,has high infection rate and disease incidence in China. We first confirmed that Chinese had B19 virus infection in 1990.Scholars indicated that B19 virus is one of the important pathogens related to some diseases in our country,such as non-immune abortion,acute aplastic anemia, acute thrombocytolytic purpura and so on.B19 virus has an icosahedral capsid that encapsidates a single-stranded DNA genome(5596 bp)which encodes a nonstructural protein(NS1),two structural proteins(VP1,VP2)and two capsid proteins(7.5 kDa and 11 kDa). VPl(83kDa)differs from VP2(58 kDa)only in a N-terminal unique region, which is composed of additional 227 amino acids with the name of VP1-unique protein(VP1u protein).B19 virus was divided into three genotypes based on its nucleotide sequence variation:typeâ… (prototype),typeâ…¡(A6-and LaLi-like)and typeâ…¢(V9-like).The variational ratio among them is 10%.We analyzed DNA sequence variation of the prevalent strain(XA strain)of B19 virus from Chinese with the assistance of the national natural science foundation(39870021),and discovered that B19-XA strain had some variations.VPlu region's variation will determine the immunologic characteristic of B19-XA strain,that because the VP1u protein mostly locates outside of the virion and can induce neutralizing antibodies.Recent study shows that VP1u protein has phospholipase A₂(PLA₂) activity,which plays an important role in the nosogenesis and development of many diseases with the character of hydrolysis fatty acids and lysophospholipids.In view of different genotypes of B19 virus,the influence put by antigen sites on diagnostic reagent and vaccine,the importance of VP1u protein in the pathopoiesia process of B19 virus,the study of B19-XA strain VP1u protein is helpful not only in prevention and treatment of B19 infection,but also in advancing the study on its nosogenesis and vaccine.Objective:The experiment aims at expressing and purifying the VP1u protein, predicting and detecting the first and secondary structures of VP1u protein by software and circular dichroism spectra.Use the VP1u protein immunize animals and detect the titre of anti-VP1u protein antibody,then carry out the pathologic study on the organs(heart,liver,lung,intestines,synovium of joint) of the immunized animals.Observe the effect that VPlu protein has on the T lymphocyte in vitro.Use the VP1u protein to construct the Enzyme linked immunosorbent assay(ELISA)system to detecte B19 virus antibody in order to reveal the immunogenicity of B19-XA initially,partly reveal the nosogenesis of B19 virus,and propel the prevention and treatment on B19.Methods: 1.To prove that pQE30-VP1 plasmid includes VP1u segment.Send the pQE30-VP1 plasmid constructed and stored in our lab to the biotech company to detect the sequence to prove that pQE30-VP1 plasmid includes VP1u segment.2.To express and purify the B19-XA VPlu protein.Ferment the E.coli M15 with pQE30-VP1 plasmid and induced in low temperature by IPTG to express fusion protein,then purify the VP1 unique protein by ion exchange column and molecular sieve column.Identify VP1u protein and its purity quotient by SDS-PAGE.3.To analyse and determine the structure of the protein.Use AnthePro 5.0 software to analyse the first structure while use the Hierarchical Neural Network method to analyse the secondary structure,and detect the secondary structure by circular dichroism spectra.4.To study the immunologic characteristic of the protein.Observe the effect that VP1u protein has on the BALB/c mice lymphocyte in vitro experiments.In vivo experiments,use the VP1u protein immunize animals and detect the titre of anti-VP1u protein antibody,then carry out the pathologic study on the organs(heart,liver,lung,intestines,synovium of joint)of the immunized animals.5.To establish ELISA method.Enzyme label plates are coated by B19-XA VPlu protein,optimize this method and compare it with polymerase chain reaction(PCR)and Parvovirus B19 ELISA Kit of Germany to evaluate the consistency.Results:1.Random coil took a larger part in the secondary structure of VP1u protein detected by Hierarchical Neural Network method and circular dichroism spectra,and they were 60.00%and 46.10%. 2.When use ELISA system to detect the titer of antibodies which are against B19-XA strain VP1u protein,the optimal concentration of coated antigen VP1u protein was between 20ng⁵0ng per well.Antibodies was still detected when the serum dilution reaches 1:16000(OD=2.04),it indicates that the immunogenicity and reactogenicity of VP1u protein were in good state.3.There were obvious pathological changing in heart,liver and synovium of joint of the immuned BABL/c mice.Under the light microscope,the heart pathological changes lay in the Henle's fissures broaden,hemangiectasis;and dissolving myofilament,Henle's fissures broaden,leukomonocyte in the broad Henle's fissures,vacuolization of the chondriosomes were shown under the electron microscopy.Under the light microscope,the liver pathological changes lay in zone of necrosis,blood sinus broaden;under the electron microscopy,the liver pathological changes lay in ecptomas on the cell,oncotic and consolidated microvillus.Under the light microscope,the synovium of joint lay in chronic inflammation changes;under the electron microscopy,we could seen more synovial cell and collogen.4.This ELISA system constructed by VPlu protein had no cross-reaction with the serum antibodies of adenovirus,respiratory syncytial virus,influenza virus,parainfluenza virus,and human herpes virus.The optimal concentration of coated antigen VP1 unique protein was 25ng per well and the optimal serum dilution was 1:200.The sensitivity was 88.37%and the specificity was 96.15% when it was used to detect B19 IgM.The ELISA method was not only in good concordance with PCR(kappa＞0.75),bur also in good coincidence with Parvovirus B19 IgM ELISA Kit(Coincidence=96.8%).And it was in good concordance with Parvovirus B19 IgGELISAKit as well(kappa＞0.75).Conclusion: 1.For the first time,the research shows that the random domain in the secondary structure of B19-XA VP1u protein took a large part,which can stimulate many kinds of antibodies(includes autoantibody)to cause pathological changes in multisystem and multiple organs.It may relate to the complicated clinial manifestation of B19 virus infection.2.BALB/c mice immunized by B19-XA strain VPlu protein can generate specific antibody.The ELISA test showed that when the serum dilution reached 1:16000,it was also positive,which explained a good antigenicity of VP1u protein.3.There were obviously pathological changing in heart,liver and synovium of joint of immuned BABL/c mice,which indicated that VP1u protein plays an important role in broad morbigenous spectrum of B19 virus.It may relate to the PLA₂ activity of VP1u protein.4.The ELISA system established to detect the antibody against B19 has the advantages of high sensitivity,strong specificity,economical and convenience.