Cloning, Expression And Antibody Preparation Of Bombyx Mori Densovirus (BmDNV-1) Structural Gene VP4

The family Parvoviridae includes two subfamilies:the Parvovirinae,members of which infect vertebrates,and the Densovirinae,members of which infect invertebrates. These viruses are nonenveloped and icosahedral,their sizes are 18～26 nm in diameter. Many parvoviruses from vertebrates package predominantly the"+" strand (complementary to mRNA),whereas most DNVs package in equal mounts both the "+" and the"-" strands in separate particles.Densovirus,Brevidensovirus,Iteravirus and Pefudensovirus are composed of a subfamily Densovirinae.Bombyx mori densovirus(BmDNV-1),based on the previously reported genome sequence,constitutes by itself a separate genus(Iteravirus) within the Densovirinae subfamily of the family parvoviridae.These virus package a linear,single tranded DNA of about 4±6 kb with terminal hairpins that are required for virus replication.It is a perfect model virus to study gene regulation because of its simple genomic structure which consisted of only structure protein and non-structure protein genes.We reported here that the structural protein gene VP4 of the BmDNV-1 was amplified by PCR and inserted into prokaryotic expression vector pMAL-c2X and then expressed in Escherichia coli DH10B strain.Nucleotide analysis showed that the VP4 gene contains 1 462 bp and encodes 487 amino acids with a predicted protein size of 54 000.The fusion protein was identified by Western-blotting with MBP-tag antibody.The expression conditions including the concentration of IPTG and the inducion time were optimized to achieve the highest expression level.The maximal expression level of the fusion protein was reached when the induction time was 3.5 hour and the concentration of IPTG was 0.7 mmol/L.The target fusion protein with molecular weight of 96 000 was induced by IPTG and purified by Amylose affinity chromatography.SDS-PAGE analysis showed that the protein purity reached above 90%.Then the purified vp4 protein was used to immunize the New Zealand rabbit to produce an antibody against this protein.The further study on the function of BmDNV-1-VP4 is underway.