PCR AMPLIFICATION AND CLONING OF ELEVEN OVERLAPPING SEGMENTS OF BIV127 env FOR BACTERIAL EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF EXPRESSED PROTEINS

Abstract

Bacterially expressed segments of bovine immunodeficiency virus (BIV) envelope were tested for immunological reactivity with sera from cows, rabbits, and a guinea pig. Overlapping segments of the gene coding for BIV127 envelope surface (SU) and transmembrane (TM) regions were generated by the polymerase chain reaction and subcloned into two fusion protein expression systems: pGEX 4-T-1, a glutathione-Stransferase (GST) fusion system (Pharmacia), and pMAL-C2, a maltose binding protein (MBP) fusion system (New England Biolabs). E. co//transformed with the recombinant plasmids were induced to express BIV-GST or BIV-MBP fusion proteins. The fusion proteins were purified by affinity chromatography using Glutathione Sepharose 4B or amylose resin, respectively. The BIV-MBP peptides were then separated by size on SDS-PAGE gels and transferred onto lmmobilon filters by semi-dry blotting. BIV-MBP purified fusion proteins were also cleaved by factor Xa protease to release the BIV fragment from its fusion partner prior to separation by SOS-PAGE gradient gels and western blot. Fusion proteins were detected using goat anti-GST antibodies or rabbit anti-MBP and tested for reactivity with sera from naturally and experimentally infected cows, an experimentally infected rabbit, immunized rabbits, and an immunized guinea pig. Additionally, sera to synthetic peptides derived from the BIV env gene were tested. Primary sera were incubated with an appropriate horseradish peroxidase-conjugated (HRP) secondary antibody using the enhanced chemi-luminescence system and exposed to x-ray film. The pGEX (GST) expression system showed low yields of BIV-GST fusion protein compared to GST controls expressed with no fusion partner. In contrast, relatively large amounts of BIV-MBP fusion proteins were expressed and could be visualized by Coomassie staining on SDS-PAGE gels as well as western blot. Analysis of the SDS-PAGE and western blot results (of BIV-MBP antigen against cow and rabbit sera) showed BIV reactivity in the BIV-MBP fusion proteins. However, with some antigens, normal control sera also reacted to the BIV-MBP fusion proteins or to copurified protein(s) of similar molecular weight. To evade "non-specific" MBP or copurified protein reactivity, BIV-MBP fusion proteins were cleaved with factor Xa and electrophoresed on 4-20% SDS-PAGE gradient gels prior to western blotting, and tested with a similar panel of sera. An immunogenic region of the amino-terminus of TM corresponding to the extracellular domain (represented by TM1-MBP) was recognized by sera from a naturally infected cow and a cow transfused with a field isolate in both the uncleaved and factor Xa cleaved experiments. Rabbit sera from a BIV127-infected rabbit and a rabbit immunized with purified BIV virion also recognized the cleavage product from factor Xa cleaved TM1-MBP antigen.