| B19 is a kind of parvovirus, which has the simplest structure compared to other animal virus and is a linear single-stranded DNA. Human parvovirus B19 was found in 1975. Parvovirus B19 is the only kind of virus causing human diseases. Many human diseases, for example, erythema infectiosum, arthritis and granulocytopenia, even the severe diseases of hematopoietic system, are due to the infection of parvovirus B19. It can be spread by respiratory tract, blood and vertical transmission between mother and baby, leading to local prevalence in community, nosocomial infection and outbreak. There exists parvovirus B19 worldwide. In 1990, it was confirmed for the first time that there exists parvovirus B19 in China. 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 andrheumatoid arthritis of children in China. At the same time, the title of the protective antibody is low in serum of women and child in our country, so that the infection of parvovirus B19 is easy to happen. Especially in those patients whose immunity are low or absent, parvovirus B19 can cause persistent infection, severe diseases of hematopoietic system and chronic anemia, even endanger human lives.The gene of parvovirus B19 is about 5.6kb, composed of one intron and two repetitive sequences at the two ends. There are two big open reading frames in the genome, which encode the nonstructure protein (NS) and the two capsid proteins (VP1 and VP2). Variations appear in the gene of parvovirus B19. NS gene is very conservative and important for the reproduction of the virus. VP1 and VP2 show relatively more variations, the rate of which is about 2-3%. Parvovirus B19 is classified into four types according to the results of enzyme cutting. The analysis of three strains of parvovirus B19 from Xi'an in China showed that they have obvious differences in both nucleic acid and amino acid compared to strains from other country and belong to the V gene type.VP1 and VP2 proteins form the capsid of parvovirus B19, coating at the surface of the virus and related to the antigenicity of it. The main epitope of neutral antigen lies in the VP1 unique area and the VP1-VP2 connective area. VP1 unique area is the main antigen stimulating body to produce neutral antibody. Non structrure protein NS is related to the virulence. VP1 and VP2 proteins, especially the VP1 uniquearea, 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 VPl 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 VPl protein, purify the protein and produce anti-VPl polyclonal antibody with certain sensitivity and specificity for the further diagnosis of parvovirus B19 of China and production of vaccine. To construct the eukaryotic expressive vector, for the further study of the biological functions of VPl protein.Methods: 1.VP1 gene was obtained from the constructed pUC19-VPl plasmid by the methods of enzyme cutting, retrieving and so on. The obtained fragment was inserted into the corresponding site of prokaryotic expressive vector pQE 30.The plasmid was evaluated by enzyme cutting. VPl-pQE 30 was cut by other two enzymes to gain VPl gene for the construction of another prokaryotic expressive plasmid VPl- pRSET A. VPl-pQE 30 and VP1-pRSET A were analysed by sequencing.2. The E. coli. with VPl-pQE 30 or VPl-pRSET A were induced to express confluenced VPl protein. SDS-PAGE wasused to analyse the expression of the protein and Wester...
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