| Human parvovirus B19 (B19) was first discovered in 1975 in England in serum of a healthy blood donor. It is the one of the two pathogenic human viruses belonging to the Parvoviridae family with a worldwide distribution. It infects human of all ages and causes several syndromes. It has been found that in various parts of the world, many women at the age of gestational are susceptible to B19 infection. When infection occurs during pregnancy it may cause severe anemia, nonimmune hydrops fetalis (NIHF), a variety of symptoms of fetal damage and fetal death. Although B19 can be detected in serum by EM, B19 antigen enzyme-linked immunosorbent assays (ELISA), and even hemagglutination, B19 virus is usually detected by isolation of viral DNA by direct hybridization or by the polymerase chain reaction (PCR).In present study, we used nested-PCR which is more sensitive than single round PCR to detect the B19 viral DNA from about 1700 cases of general patients, including 900 women and 800 common adults (men and women). The positive detective rates were 8.33% in group I (women) and 4.50% in group II (common adults, men and women). Our data demonstrated that prevalence of human parvovirus B19 infection in childbearing- age women was much higher than that in ordinary population and diagnosis of B19 virus in pregnant women should be adopted to prevent newborns infection.The B19 contains a single stranded, linear DNA genome whose size is approximately 5.6kb. The genome encodes a non-structural protein, NS1 and two structural protein that comprise the B19 capsid, VP1 (84kDa) and VP2 (58kDa), which are identical with the exception of 227 amino acids at the amino-terminal end of the VP1-protein, so-called VP1-unique region (Vp1u). A conserved motif (HDXXY) with phospholipase A2 activity has recently been identified in the N-terminal unique region of the VP1 capsid protein in approximately 30 different parvoviruses, including B19. PLA2 has become a potential target for anti drug design. However, there is little known about the molecular biology of this virus. Therefore, it is essential to characterize the viral PLA2 of the B19 to further understand the mechanism and function of this enzyme.In this study, we changed several key amino acids in the PLA2 domain of B19 by the site-directed mutagenesis, sPLA2 activity for both the wilt type and mutants will be assayed and compared. Briefly, the VP1u containing the PLA2 domain was ligated with PUC18a plasmid, the critical amino acids were mutated by the method of site-directed mutagenesis. Then the mutated VP1u was cut out and ligated with an expression vector pMal-c2x to obtain the plasmid pMal-uVP1. Three key amino acids were mutated by this method. The expressed proteins with PLA2 were analysed by the SDS-PAGE and Western Blot to evaluate the expression level and specificity. Then the target proteins were purified by affinity column and the PLA2 activity assay will be performed... |
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