Sequence Analysis, Molecular Cloning And Expression Of Human Astrovirus Serotype 1 Capsid Protein And Preparation Of Polyclonal Antibodies In Rabbits

Human astrovirus (HAstV) are detected as a common cause of viral acute gastroenteritis. Eight astrovirus serotypes have been described among human astroviruses (HAstV 1-8), with Human astrovirus serotype 1 (HAstV-1) being the most prevalent worldwide.The genome of HAstV-1 is composed of an approximately 6.8 kilobases (kb) nonenveloped single-stranded positive-sense RNA molecule, organized in three open reading frames (ORFs):ORFs 1a, 1b and 2. Recently, Bioinformatic predictions revealed that one of the main structural proteins which are encoded by ORF2 of HAstV-1 could be involved in virus-cell interaction and it seems to contain neutralizing and immunoreactive epitopes.In this study, comparison of the capsid protein precursors encoded by ORF2 of 11 strains of HAstV-1 was carried out. A phylogenetic tree showed two genogroups of HAstV-1 that corresponded exactly to the regains. The structure and properties about encoding protein of VP26 were analyzed and predicted by bioinformatics, as well as the immunological characteristics.Afterwards, we cloned and expressed in Escherichia coli (E.coli) of a recombinant VP26 (rVP26) protein, followed by purification by NTA-Ni2+ agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE),bicinchoninic acid assay (BCA assay) and Western blot (WB). Furthermore, we immuned the rabbits for 4 times. The serum of rabbits was extracted from heart as polyclonal antibody. The titer and specificity of rabbit's antiserum was measured by ELISA.The results show that prokaryotic expression vector pET30a(+)-VP26 was successfully constructed. The rVP26 protein could be highly expressed in E.coli (Rosetta 2) cells. The titer of the antiserum measured by ELISA could achieve 1:8000. This indicated that antibody and purified recombinant protein had a good reaction and high titer. They could meet the experimental require.In conclusion, (1) rVP26 can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies. (2)This cloning, expression and purification system can be a suitable method to obtain large amounts of highly purified rVP26 for further characterization to evaluate its utility for developing new detection methods and vaccines.(3)It also could be more useful than complete viral particles, which are commonly used in immunoenzyme techniques as the coating antigen in commercially available ELISA kits used to detect antibodies.(4)The polyclonal antibodies of rVP26 could meet the experimental require of ELISA.To sum up, the establishment of prokaryotic expression system and preparation of poly-clonal antibodies could be references for the future studies. They will be helpful for further studying the pathogenesis of HAstV-1 infection and development of HAstV-1 vaccines as the immunological tools.