The Construction Of Eukaryotic Expression Vector Of The NSP4 Gene Of Porcine Group A Rotavirus And The Comparison Of Immunodetection

Rotavirus infection is the foremost cause of severe gastroenteritis of young children and piglets worldwide. Rotavirus gastroenteritis is characterized by watery diarrhea usually prec- eded or accompanied by vomiting and fever. Rotavirus infection can be fatal as a result of severe dehydration, electrolyte loss,and acidosis. Rotavirus is severely threaten to raise pigs, which is prevented by means of vaccines. Efforts to develop safe and effective vaccines became the most important task. Early studies focused on serum antibody responses to different G(VP7) serotypes,as measured by neutralization assays, and suggested that serotype cross-reactive immunity plays an important role in protection. Whether neutralizing antibody responses to outer capsid proteins VP4 and VP7 play a critical role in protective immunity against rotavirus-associated diarrhea remains controversial.The role of nonstructural proteins in the induction of protective immunity has not been extensively studied in rotavirus infections, but it has recently emerged from some studies.NSP4, a rotavirus nonstructural glycoprotein, plays a role in rotavirus morphogenesis and is the viral enterotoxin capable of inducing secretory diarrhea in infant mice. Sequence analyses of the rotavirus entero- toxin NSP4 from humans and animals have revealed the existence of six (A to F) distinct NSP4 genotypes.The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells.In order to express NSP4 in eukaryotic cells,the gene encoding NSP4 from rotavirus strain Wa was first cloned into the clonal vector pMD-18T as a KpnI-BamHI fragment to insert in eukaryotic expression vector PVXI, incorporated in the parent vector. Plasmids were also successfully constructed that expressed the protein of NSP4. The detail steps is the fowlling:First,These were generated using standard PCR techniques employing oligonucleotide primers to insert a Ndel restriction site/AUG start codon and BamHI restriction site/stop codon at various positions within the NSP4 coding sequence. Second,the porcine rotavirus(RV) was propagated in cell culture,and harvested. Double-stranded RNA extracted from the virus...