We have attempted to express rotavirus antigen in E. coli as a means of developing an effective and safe rotavirus vaccine. If such an antigen is located on the bacterial surface, it may stimulate local immunity by colonizing the small intestine. Toward this goal, we have utilized an open reading frame (ORF) expression vector (pORF2) which may direct the expression of rotavirus gene segments. The insertion of rotavirus cDNA sequences in this vector may allow the expression of hybrid proteins which could be transported to the cell surface. When we cloned various sets of Sau 3A partial digests of NCDV and RRV gene (encoding VP7) cDNA into pORF2, only a few of these constructs expressed recombinant molecules, although the in-frame insertion of the gene segments into pORF2 had been achieved. The highest levels of expression (up to 14% of E. coli protein) were achieved with the shorter segments; however, the resulting hybrid proteins tested by immunoprecipitation were not recognized by either polyclonal or monoclonal antisera. When we cloned larger fragments of the NCDV VP7 gene, the level of expression was not high enough to allow further studies with this system. We also made constructs in which a lambda PL promoter fragment was introduced 5' upstream of the fusion genes instead of ompF promoter originally provided with pORF2 vector. The level of expression achieved with this stronger promoter was, however, not significantly increased. The PL promoter has also been used to attempt expression of defined segments of the NCDV gene 9 (VP7 gene). One such construct (carrying 822bp coding sequence) directed the expression of a protein of +28,000 daltons when transformed into E. coli. Immunological examination of this protein is in progress.
