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ASL of human parvovirus B19 activity affect
B19 virus 25nm in diameter nonenveloped capsid was icosahedron structure, genomic single-stranded linear DNA, the total length of about 5.6kb left end of the genome of non-structural protein NS1 coding region of the genome right end of the structural proteins of VP1 ( 84kDa) and VP2 (58 kDa) coding region encoding VP2 gene sequence is entirely contained in the VP1 sequence, the N-terminus of the VP1 than VP2 227 amino acids, called the VP1 unique region (uVP1). The study found that most of the parvovirus VP1 unique region with phospholipase A2 activity, and parvovirus B19 also has this feature.ASL activity of VP1 unique region protein in the process of virus infection of host cells, have to make the virus able to dissolve the host cell membrane, and thus play an important role in the process of viral infection, the region has become one of the antiviral drug design a potential target.
In this study, site-directed mutagenesis by a will uVP1 of key amino acids ASL activity and virus infection, both in terms of VP1 unique area features the focus of the study is unique on the acyl-CoA binding domain containing 6 plays a key role in the 130,132134,154 bits of amino acids. In the experiment, the first purpose of gene VP1u inserted into the PUC-18a vector, and then to construct the recombinant plasmid PUC-uVP1 as a template, the use of PCR for rapid mutation, to get different sites of mutation after cloning the PUC-muVP1, were sequenced to confirm is correct, then mutation of unique region inserted into the expression vector pMal-c2x to construct recombinant prokaryotic expression vector pMal-umVP1 The. Respectively, after the mutation of the different sites of the prokaryotic expression vector induced expression in E. coli by SDS-PAGE and western-blot analysis detected, and then by expanding the training induced expression, and column purification, the collection of the target protein and to detect its ASL activity . This experiment to get the 154 mutation uVP1 activity tests showed that the 154 mutation uVP1 the enzyme activity is completely lost, suggesting that the 154 aspartic acid essential to the maintenance of the ASL activity.Meanwhile, the infectious clone of the building after the mutation of the corresponding sites of the B19 genome, provide a basic tool for uVP1 the function at the cellular level.The genome 2251 ~ 4291bp of gene sequences cloned in the process of building a mutation infection sexual cloning of pBluescript II of KS (+) built reorganization of plasmid PB2040 mutation PCR of the template, with the corresponding mutations lead material in vitro rapid mutagenesis carried out mutations, screening the mutation was cloned and sequenced correctly, the 2040bp fragment was cloned mutations after missing 2040bp fragment B19-4 244 plasmid in order to build up the appropriate mutant clones B19-4244m. The experiments have been successfully constructed pB2040, and by PCR-SDM, 132,134 and 154 amino acids of mutant pBG132R, pBG134R pBD154A, an intermediate for the construction of mutant infectious clone. These results further study the function of VP1 unique region provides the basic molecular biology tools to clarify the parvovirus B19 infection mechanism provides a theoretical basis.
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