Cloning, Expression And Purification Of Human Bocavirus (hbov) Structural Protein Gene And The Expression Of Vp1 In Baculovirus System

Lower respiratoty tract infection (LRTI) is the most common disease of children, and it is a leading cause for the mortality of children. In October of 2005, a new parvovirus was first detected by Allander from nasopharyngeal aspirates of children with lower respiratoty infection, it is a pathogen of bronchiolitis and also can induce upper or lower respiratoty infection of children, even severe respiratoty disease. Studies from its molecular biology, analysis of phylogenetic tree and epidemiology indicated that this new virus has basic characteristics of the family Parvoviridae and shares the similar structural feature with the members of the other known bocavirus genus of subfamily Parvovirinae, suggesting that this virus belongs to a new member of the Genus Bocavirus, named Human Bocavirus (HBoV). Nowadays, the genus Bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and the recently identified human bocavirus (HBoV).HBoV is a non-enveloped single-stranded DNA virus, the current genomic DNA reference sequence of HBoV is 5299 nt in length and it is the second one of parvoviruses which cause human disease after human parvovirus B19. Studies from all over world showed that HBoV infection generally existed in children with respiratory tract illnesses and were also detected in serum, fecal and urine samples, indicating that the virus was related with gastrointestinal tract disease. HBoV alone can infect people and also co-infect people with other viruses. HBoV infection occurs through the whole year, but it usually occurs in the cold season, such as late autumn, winter and early spring. At present, the biological features of HBoV and its significance of human disease by this virus remain unknown.In this study, the forward and reverse primers were designed based on the reported structural protein gene VP1and VP2 of HBoV genome isolated by our laboratory (Accession number of GeneBank:GU 139423). These two genes were amplified by using PCR and inserted into prokaryotic expression vector pMAL-c2x and pET28a and then transformed into the Escherichia coli DH5a strain and BL21(DE3), respectively. The fusion protein expression was induced by IPTG and identified by Western Blot with MBP-tag antibody and His-tag antibody. The target fusion protein was then purified by Amylose affinity chromatography and Talon Resin Column. The purified VP2 protein was used to immunize the New Zealand rabbit to prepare an antibody against this protein.To find out whether the VP1 unique part of the VP1 protein is expressed alone or cleaved from the VP1 protein, the full length capsid protein VPlgene was inserted into the baculovirus expression transfer vector (pFastBacHTA) to obtain the recombinant Bacmid DNA which was then transfected into insect cell Sf9.7-10 days post transfection, the supernatant of transfected-cells was used to infect Sf9 cells to get more viruses with higher titer. The recombinant VP1 protein was identified by Western Blot with His-tag antibody or hyper-immune serum against VP1-U of HBoV. Our results showed that no VP1 unique part of VP1 protein was produced from the recombinant baculovirus.