Expression And Immunological Properties Of Rotavirus Nonstructural Protein NSP3

Rotavirus, a member of the family Reoviridae, is the most common cause of severe diarrhea in children worldwide, which is responsible for an estimated 611,000 deaths every year. The rotavirus genome, surrounded by a three-layered virion and composed of 11 segments of double-stranded RNA, codes for six structural (VP) and six nonstructural (NSP) proteins. Cross-reacting antigens have permitted serological classification of seven groups (A-G). Only groups A, B, and C infect humans, with group A causing the vast majority of childhood diarrhea. Group A NSP3 is a homodimeric 36 kDa protein, with its N-terminal (NSP3-N) portion binding to a group-specific tetra-nucleotide consensus sequence found at the 3' ends of rotavirus mRNA and its C-terminal domain (NSP3-C) competing with PABP for the same segment of eIF4G: NSP3 shuts off host cell protein synthesis and is thought to play a critical role in genome replication and morphogenesis of rotavirus.The gene of NSP3 cloned from rotavirus strain TB-Chen isolated from a clinic sample was inserted into the expression vector pETL, and the recombinant plasmid pET-NSP3 was constructed. The recombinant NSP3 was highly expressed in E.coli BL21 (DE3) cells, which accounted for 28.6% of total protein. This protein was purified and inoculated into guinea pigs to produce antiserum. It was shown by Western blot that the recombinant NSP3 had remarkable immunoreactivity and could react with its antiserum specifically. Immunofluorescence assay showed that NSP3 was distributed throughout the cytoplasm with distinct patterns, some was distributed in a homogeneous diffuse pattern, others was distributed in a punctuated pattern and was more abundant closer to the perinuclear region. These results laid the foundation of further study on the structure, function and immunological properties ofthe NSP3 of this strain rotavirus.