The Preliminary Analysis Of The Function Of Structural Protein And Non-structural Protein Of Human Bocavirus

Swedish scientist Tobias Allander discovered a new parvovirus in October2005from nosapharingeal aspirates of children associated with lower respiratory disease and this new parvovirus is named the Human Bocavirus (HBoV) whose genome is a single-stranded linear DNA (diameter ranges from18-25nm) about5300nucleotides in length included in an nonenveloped particle.These viruses have a T=l icosaheral protein capsid coat which protects from inactivation by moderate PH changes, high temperatures, and high salt concentration. The symptoms including fever, cough, rhinitis and, more rarely, breathting difficult or rashes were observed in HBoV-infected individuals. HBoV is classified in the genus Bocavirus within the family Parvoviridae.HBoV contains two open reading frames. One of the ORFs codes two nonstructural proteins, and the other encodes at least two capsid proteins (VP1and VP2). VP1-unique region (VP1u) possesses a phospholipase A2-like activity (PLA2). This viral PLA2has been shown to be of crucial importance for parvoviral infectivity. Some data show that the gene coding nonstructural proteins is conservative, wheares, the gene coding capsid proteins is unstable.There is a repeat sequence (Inverted terminal repeat, ITR) at3’and5’ terminal of HBoV DNA respectively. The ITR can form Palindromes structure that servses as a primer when the DNA of parvovirus replicates.The VP1(4160-4480nt) gene of the HBoV was amplified by PCR and inserted into prokaryotic expression vector pET-49b(+).The fusion protein fused with His-tag was induced by IPTG and purified by infility purification system.Then the purified protein was cutted off from the fusion protein to remove the His-tag, grinded, and dissolved with PBS. Finally the purified fusion protein was used to immunize the mice to prepare the corresponding antibody. This study provides a reliable tool for further investigation of transcription and translation mechanisms of this gene.At the same time, we studied the localization of capsid proteins (VP1and VP2) and nonstructural proteins (NS1) in the293T cells. These genes were amplified by PCR and inserted into eukaryotic expression vector pEGFP. Then these recombinant plasmids were transfected into293T cells, the localization of capsid proteins (VP1and VP2) and nonstructural proteins (NS1) in the293T cells were observed with Fluorescence microscope. In addition, we found that apoptosis occured in HeLa cells transfected with the plasmid which expressed the nonstructural proteins (NP1). These results were confirmed by DAPI staining and caspase assay.