Antibody Preparation And Activity Analysis Of The Phospholipase A2 Of The VP1 Unique Part Of Human Parvovirus B19

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.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 proteins comprising 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 part (Vplu). A conserved motif (HDXXY and YXGXG) with phospholipase A2 activity has recently been identified in the N-terminal unique region of the VP1 capsid protein in approximately 34 different parvoviruses, including B19. PLA2 is essential for the replication and infectivity of parvoviruses and is a potential target for anti drug design against parvovirus infection. 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, and vPLA2 activity for both the wild type and mutants were assayed and compared.Firstly, the structural protein gene vplu of the B19 was amplified by PCR and inserted into prokaryotic expression vector pMAL-c2x and then expressed in Escherichia coli DH5a strain. The expression conditions including the concentration of IPTG and the inducion time were optimized to achieve the highest expression level. Then, the target fusion protein was purified by Amylose affinity chromatography and the Tag-MBP protein was cleaved from the fusion protein by Factor Xa. Finally, the purified vplu protein was used to immunize the New Zealand rabbit to produce an antibody against this protein.Secondly, based on the construct of PUC-VPlu plasmid, the critical amino acids were mutated by the method of site-directed mutagenesis. Then the mutated VPlu was cut out and inserted into an expression vector pMal-c2x to obtain the plasmid pMal-mVPlu. Three key amino acids 133,175,195 consisting of a catalytic center of the PLA2 were mutated by this method. Then the target proteins were purified by affinity column and the PLA2 activity assay was performed as described by manufacturer's manual. Our data showed that B19 Vplu indeed exhibited a PLA2 activity as demonstrated by all the parvoviruses and the PLA2 activity was reduced dramatically by mutation of key amino acids in the position of 133 and 175, but a slight increase by mutation of key amino acid of 195. Further studies are necessary to elucide the mechanism of PLA2 in the replication of B19.