UBC Theses and Dissertations
CBD-cellulose : a novel adjuvant system Tekant, Bahar
Glycoprotein D gene of Herpes Simplex Virus Type 1 (gD.1) was cloned in Escherichia coli as a gene fusion to the endoglucanase A cellulose binding domain (CBD) gene of Cellulomonas fimi. The initial construct produced a form of CBDCenA-gD.1 fusion protein which severely inhibited the growth of E. coli. The inhibitory effect was attributed to the presence of the transmembrane region of the gD.1 protein. A cassette plasmid was constructed to express CBDCenA fused in frame to any other gene. A truncated gD.1 gene lacking the coding regions for the leader peptide and the transmembrane/cytoplasmic domains was inserted into this cassette, and the synthesis of 45.9 kDa fusion protein was shown. The fusion protein was severely degraded, and also, associated with the cell membranes. CBDCenA-PhoA, a chimeric protein containing alkaline phosphatase, was used to investigate the potential adjuvant effect of immunization with a CBD-fusion protein bound to cellulose. Immunogens tested included alkaline phosphatase alone, CBDCenA-PhoA alone, alkaline phosphatase in Avicel (micro crystalline cellulose) solution, CBDCenA-Pho A-Alum (aluminum hydroxide) and CBDCenA-PhoA-Avicel. Mice immunized with CBDCenA-PhoA-Avicel showed the highest anti-alkaline phosphatase and anti-CenA antibody titers. Animals immunized with alkaline phosphatase-Avicel, CBDCenA-PhoA-Alum, CBDCenA-PhoA or alkaline phosphatase alone showed mean secondary anti-alkaline phosphatase antibody titers that were 29%, 26%, 17% and 17% of the CBDCenAPhoA-Avicel titers, respectively. Animals immunized with CBDCenA-PhoA-Alum or CBDCenA-PhoA alone showed mean secondary anti-CenA antibody titers that were 57% and 42% of the CBDCenA-PhoA-Avicel titers, respectively. The results indicate that the use of CBD fusions to present an antigen bound to cellulose provides an effective adjuvant for antibody responses.
Item Citations and Data