Expression, Purification And Study Of Immunogenicity Of Human Parvovirus B19 VP1 Unique Protein

Human parvovirus B19(PVB19),the only parvovirus known to be pathogenic parvoviridae in human,belongs to the genus parvovirus in the family. It is the smallest animal DNA virus. PVB19 infection often results in a variety of clinical manifestations in adults and children . B19 has the simplest structure compared to other animal virus and is a linear single-stranded DNA.Human parvovirus B19 was found in 1975. it was confirmed for the first time that there exists parvovirus B19 in China In 1990.Followed studies found it is one of the important etiological factors of non-immunity abortion of pregnant women,acute aplastic crisis of children and acute idiopathic thrombocytopenic purpura and related closely to congenital heart diseases and rheumatoid arthritis of children in China.The genome of B19 consists of DNA of approximately 5.6kb.The genome encodes a non-structural protein, NS1, and two structural proteins that comprise the B19 capsid, the minor structural protein, VPl, and major structural protein, VP2. B19 capsids are composed mainly of VP2 protein, VP1 protein contributes less than 10% of capsid protein, and its specific region is probably exposed on virion surface. Most viral neutralizing epitopes map in the VP1 specific and VP1-VP2 junction regions. Only proteins encoded by B19 VP1 specific and VP1-VP2 junction regions can stimulate bodies to produce protective antibody. NS gene is very conservative and important for the reproduction of the virus and is related to the virulence.VP1 and VP2 proteins,especially the VPl unique area, are significant in the diagnosis and production of the protective vaccine.However, parvovirus B19 can not be cultured in traditional bases,which makes it difficult to get antigen. For another,strains of parvovirus B19 from China (XA strains) have obvious differences in both nucleic acid and amino acid compared to the standard strains.It is necessary to develop great amount of VP1 protein of the strains of parvovirus B19 from China (XA strains)for the production of diagnostic reagent and vaccine.Objective:To construct the prokaryotic expressive vector, express great mount of VP1 protein , purify the protein and produce anti-VP1 monoclonal antibody with certain sensitivity and specificity for the further diagnosis of parvovirus B19 of China and production of vaccine and for the further study of the biological functions of VP1 protein.Methods:1. VPl gene was obtained from the constructed VP1-PUC19 plasmid stored in my lab by the methods of enzyme cutting,retrieving and so on.The obtained fragment was inserted into the corresponding site of prokaryotic expressive vector PQE30.The plasmid was evaluated by enzyme cutting.VP1-PQE30 was transformed into E.coli M15 and was analysed by sequencing. 2. The E.coli M15 with VP1-PQE30 were fermented and induced by IPTG to express confluenced VPl protein.SDS-PAGE was used to analyse the expression of the protein and Western blot was used to evaluate the expressed protein. The express confluenced VPl protein was purified by AKTA explorer 100 system.3.Female BALB/c mice were immunized with the purified VP1 protein. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method and the hybridomas were selected in HAT medium.The hybridoma cells secreting specific antibody were detected by ELISA, and the specificity of anti-VP1 monoclonal antibody(mAb)were detected by Western blot.Results:1. VP1-PUC19 was cut by BamH I and SalI,it has a segment of 480bp,consistent with expected results.The sequencing of the recombinant was confirmed correct.2. The bacterium with recombinant expressive vectors can express protein of 22KD after induction.Western blot showed it can combined with anti-6-His antibody, so it was confluenced VP1 protein.3. The bacterium induced by IPTG during fermentation can highly express B19 VP1 confluenced protein which occupied 95% in totle protein after purified by AKTA explorer 100 system.4. Two hybridoma cell strains designated as LI and ZH which had good sensitivity and specificity against VPl unique protein were obtained. Western blot showed that the antibodies were specific for VPl protein.Conclutions:1.The prokaryotic expressive system VP1-PQE30-M15 was constructed successfully .2. The E.coli M15 with VP1-PQE30 were fermented and induced by IPTG can express confluenced VPl protein.3.The express VPl protein purified by AKTA explorer 100 system was occupied 95% in totle protein.4.We obtained two clones of hybridomas stably secereting mAb against B19 VP1 protein first in China which may lay the foundation for further research genovariation of B19 VP1 and set up the dectect Kit of B19.