Plasmid for the direct subcloning from lambda gt11 to produce an LT-B fusion protein.

We wished to study LT-B fusion proteins for potential as vaccines. We reasoned that a gene that was expressed as a lambda gt11 fusion protein would have a better chance of being expressed fused to LT-B. To facilitate such constructions, we modified plasmid pYA3081. A BamHI-EcoRI-BamHI* (* is to stop codon) adapter was inserted into the single BamHI site to form a new plasmid-designated pBEB. The insert was designed so that the EcoRI site at the downstream end of the LT-B gene was in the same reading frame as the EcoRI site in the beta-galactosidase gene of lambda gt11 and termination codons occurred downstream in all three reading frames. A clone was selected from a gt11 library based on the production of a protein that reacted with antibodies to flagellin from Campylobacter jejuni. DNA from this clone was digested with EcoRI, the insert fragment purified and ligated into the EcoRI site of pBEB. A fusion protein of 43 kDa was produced that reacted in Western blot with antibodies against LT-B and flagellin. We have succeeded in making an easy method for subcloning fragments from gt11 into a vector for making LT-B fusion proteins.