A major goal of our laboratory is the development of a subunit vaccine against the ciliate, Ichthyophthirius multifiliis, an important protozoan pathogen of freshwater fish. While the disease caused by I. multifiliis (Ich) is usually lethal, fish surviving epizootics are resistant to subsequent challenge. We have identified a class of putative protective surface antigens called immobilization antigens (i-antigens). Sequence analysis of cDNA and genomic DNA encoding the i-antigens reveals that Ichthyophthirius has an anomalous genetic code such that commonly recognized "stop" codons, TAA and TAG, code for the amino acid glutamine instead. Thus, expression of Ich genes using conventional bacterial systems results in the production of truncated proteins.

Through the methods of PCR-based mutagenesis and serial overlap extension, we are attempting to biochemically alter eight TAA and TAG stop codons occurring within the first 419 nucleotides of the cDNA that encodes the 48 kd i-antigen of I. multifiliis (strain G1). These altered codons will then read as glutamine codons CAA and CAG, respectively, when cloned into bacterial cells for expression. Pair-wise series of reactions were performed in which oligonucleotide primers with the eight mismatched bases served as forward and reverse primers for each PCR reaction. Next, individual mutated and amplified products were pieced together in a second set of PCR reactions using overlap extension (ref. Horton, et. al. (1990), Biotech 8, 528). A 430 bp product is currently being sequenced to confirm presence of the eight point mutations. The mutated product will be cloned into the plasmid vector pProEx-1 for ultimate large-scale expression of the recombinant protein i-antigen.

Figure 1

Figure 2

A PCR-based technique known as overlap extension was used to modify UAA and UAG "stop" codons in the Ich i-antigen cDNA to create a template that should be readable by the translational machinery of E. coli. The schematic diagram above demonstrates the steps used to alter the first 8 UAA and UAG codons in the 1.2kb i-antigen cDNA. Initially (top line), pairs of forward and reverse PCR primers (A-H, small arrows) were used to amplify 4 separate regions spanning the first 419bp of the 1.2kb cDNA. The primers used in these reactions had mismatched bases which resulted in replacement of UAAs and UAGs with CAAs and CAGs in the amplified products. The A/B and C/D products were combined and used as templates for a second round PCR reaction (with primers A and D) to make the A/D fragment. A parallel reaction was also carried out with the E/F and G/H products to make the E/H fragment. Finally, the A/D and E/H products were combined in a third round of PCR reactions (using primers A and H) to make the 430bp fragment. The products of the individual PCR reactions are shown on the ethidium-stained agarose gel (bottom). The leftmost lane shows a l00bp DNA ladder for estimating product size (lowest band is 100bp). Similar reactions will eventually be carried out to modify the entire 1.2 kb i-antigen cDNA containing a total of 28 base pair changes.