Study On Molecular Epidemiology And Serologically Defined Antigen Of Human Bocavirus

Respiratory tract infection has its 90% origins in virus among children and youngs. These viruses which induce respiratory tract infection range over 200 types of more than 10 categories of 7 families. Heikkinen T and Jarvinen A(2003) considered that unknomn respiratory viruses are about 1/4 in all. In the last few year, there have been outbreak of virus respiratory tract infection frequently. Viruses known are mutating, and new virus are generate. Study on molecular epidemiology of respiratory viruses and identification of new virus species are key issues for the study of infectious disease.In this research, several new respiratory viruses (NL63, HKU-1, hMPV, HBoV) and major respiratory viruses were investigated in molecular epidemiology, and a VP1-U protein was expressed and purified. The goal of this research is a flow or progress of work for disease control and prevention, which was realized by reseach on prevalent, variation and distributing of respiratory viruses and on serological diagnosis.1. molecular epidemiology of HBoV1) Creation of detectable method by molecular biologyWhole virus nucleic acid were extracted from NPA samples of children who were hospitalized because of respiratory tract infection and RT, the interested conservative sequences were augmented by PCR. If the longth of electrophoresis band was in accord with interested sequence, it was thought that interested virus was in the sample.2)investigation of epidemiology of HBoVResearch on epidemiology of HBoV in given group showed that bronchiolitis, pneumonia, bronchopneumonia probably were linked to hBoV, and incidence was about 8.3%.3) Cloning and Phylogenetic Analysis of Human Bocavirus VP1 geneGenomes of 10 human bocavirus(HBoV) were amplified by PCR, cloned into pGEM-T vectors reparately, and sequenced. In this study, the full sequences and VP1 sequence of HBoV were aligned with that of 14 representative of Parvoviruses length in Parvovirinae. Phylogenetic analysis showed that HBoV capsid sequence positioned closely to B19 parvovirus, although they positioned far in phylogenetic analysis based on full sequences. Many similarities were found between HBoV and B19 in capsid by alignment on Secondary structural elements. Because both the B19 and HBoV are the only Parvoviruses which infected humankind, so study about HBoV may use for reference for B19 which had been studied about thirty years. By analysis of mutational sites, HBoV capsid protein showed a highly conserved secondary structural elements, but a highly active in VP1-U, leading end of VP2 and insertions between the strands of theβG-H. This cued that HBoV inclined to immune evasion and infectant adaptive faculty.2. Expression and purification of VP1-U protein1)VP1-U was expressed and purified in E.coli BL21(DE3)At first, antigenicity of VP1 gene was predicted. Then, the active antigenicity sequence was inserted into the pET30a(+) and expressed. The protein exclusion body was released from E.coli by the methods of ultrasonic. By several purify steps including extract with organic solvent, desalination, dilution renaturation and reverse phase chromatography.