ABSTRACT
Background: Rotavirus group A [RVA] is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 [NSP4] is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals
Objectives: The aim of this study is the evaluation of rotavirus ANSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein
Materials and Methods: Splicing by overlap extension [SOEing] PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length [FL-NSP4] and the spliced [S-NSP4] cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 [DE3] by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test
Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable
Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain [S-NSP4]. SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future