Expression of nonstructural glycoprotein NSP4 of SA11 Simian rotavirus in escherichia coli and production of antibody aginst it

ABSTRACT

Rotavirus non structural glycoprotein NSP4 can induce diarrhea in newborn mice. It has been suggested that NSP4 may be a key determinant for rotavirus pathogenesis and a target for vaccine development. In order to study the biological and morphological role of NSP4 a large amount of the purified protein and antibody against it are required. Simian rotavirus SA11 was propagated in BSCI cell, purified on cesium chloride gradients, and its genomic RNA was extracted. A cDNA from RNA segment 10 was synthesized and amplified by RT-PCR. cDNA fragment was cloned into plasmid vectors. The recombinant plasmid was characterized by restriction enzyme and sequencing. Construction of expression plasmid containing nsp4 gene was performed and expression of NSP4 was demonstrated by SDS-PAGE, Western blot, ELISA and IF using polyclonal antibody against NSP4 from SA11 infected BSC1 cells. A polyclonal antiserum against purified recombinant NSP4 was raised in rabbit; which was reacted with NSP4 in BSCI cells infected with SA11 rotavirus. These results indicated successful expression of the full-length NSP4 in E.coli to produce antibody against it and to study its biological activities