Expression And Antibody Preparation Of Structural Protein VP1u, Non-structural Protein ORFx And NP1 Of Human Bocavirus

Swedish scientist Tobias Allander and his co-workers discovered a new parvovirus in October 2005 from nosapharingeal aspirates of children associated with lower respiratory disease and the new parvovirus is named the Human Bocavirus (HBoV) whose genome is a single-stranded linear DNA about 5299 nucleotides in length included in an nonenveloped particle. HBoV is classified in the genus Bocavirus within the family Parvoviridae.HBoV contains two open reading frames which are similar to other known parvoviruses. One of the ORFs encodes at least two capsid proteins (VP1 and VP2), and the other codes nonstructural proteins (NS1) for HBoV also contains another open reading frame within the ORF of structural protein and its function is unknown.Non-structural protein NS1 is first transcribed and translated in the virus infected cells and then binds to the viral replication origin to initiate the replication of virus DNA. The virus sequence analysis indicated that there is an unknown protein ORFx and no its homologous gene was found in the parvovirus family. The Cratch Protein Predictor and the ProtFun analysis reveal that the NP1 protein does not show the catalytic function but may be related to the translation process of viral genes [3]. In order to better understand the function of non-structural protein genes ORFx, NP1, and structural protein genes VP1u, we cloned these genes, expressed them in prokaryotic expression system and then prepared polyclonal antibodies, respectively.In this study, we designed primers of ORFx, VP1u and NP1 genes based on our published sequence of the HBoV genome (GeneBank, accession number:GU 139423) and cloned these genes into the prokaryotic expression vector pMAL-c2X. The fusion proteins were induced by IPTG and purified by Amylose affinity chromatography, Then the fusion protein MBP-ORFx and MBP-NP1 were digested by enzyme Factor Xa. Finally, the fusion protein MBP-VP1u and Factor Xa-cut MBP-ORFx protein were used to immunize rabbits and factor Xa-cut MBP-NP1 protein to immunize mice. These polyclonal antibodies provide us with some reliable tools to further study the features of the non-structural protein genes ORFx, NP1 and structural protein VP1u of the HBoV.