Group A Rotavirus Nsp2 Gene Cloning, Expression And Immunological Properties

Rotavirus, as a member of the Reoviridae family, has a triple-layered capsid without envelope, approximately 70 nm in diameter. Rotavirus was recognized as the primary aetiological agent leading to nonbacterial childhood gastroenteritis in 1973, and which is estimated to be responsible for approximately 600 000 deaths due to acute gastrointestinal disease in young children throughout the world each year. Rotavirus genome comprises 11 segments of double-stranded RNA, which encodes 6 structural proteins and 6 non-structural proteins. According to the antigenic specificity of the VP6 protein, which constitutes the internal capsid, the RV strains are classified into seven groups (A to G), and in which group A rotaviruses are the major cause of acute gastroenteritis in infants and young children.Usually, NSP2 of group A rotavirus contains 317 amino acids and is a highly basic protein of relative molecular mass M_t=35,000. Between the N and C-terminal domains is a deep electropositive cleft containing a catalytic site that hydrolyzes theγ-βphosphoanhydride bond of any NTP. NSP2 plays a role in rotavirus replication by interacting with other proteins of itself, such as NSP5, to form a membrane-free viroplasm scaffold in which the segmented double-stranded RNA of the virus is replicated and packaged into previrion core particles. In addition, enzymatic analysis revealed that recombinant NSP2 possessed nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg²⁺ catalyzed hydrolysis of each of the four NTPs to NDPs with equal efficiency, indicating hydrolysis of NTP resulted in the covalent linkage of theγ-phosphate to rNSP2.TB-Chen strain was the first strain of group A human rotaviruses isolated in patient of Chinese origin that the full genome was cloned and characterized. To deeply investigate NSP2 characteristics, the whole coding gene of the NSP2 of strain TB-Chen was cloned and constructed into the recombinant plasmid pETL and expressed in a prokaryotic expression system, and intracellular distribution and immunological characteristics of this protein was further studied. The results showed that the recombinant NSP2 protein (rNSP2) could be highly expressed in E. coli cells, the expressed product accounts for 44.54% of total bacterial proteins. The rNSP2 could elicit specific antibodies in guinea pigs and these antibodies could react with the recombinant protein itself expressed in E. coli cells and NSP2 proteins synthesized in SA11- or Wa-infected MA104 cells, by immunofluoresence assay and western blot. These results lay the foundation for further study on the structure and function, immunological properties and practical use of the NSP2.