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.
Meinersmann
R J
RJ
USDA, Agricultural Research Service, Russell Research Center, Athens, GA 30613.
Khoury
C A
CA
D001427:Bacterial Toxins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D016123:Campylobacter jejuni / Q000235:genetics; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics*; D029968:Escherichia coli Proteins; D005408:Flagellin / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D005408:Flagellin; C027538:heat-labile enterotoxin, E coli; D015246:Deoxyribonuclease EcoRI; D001616:beta-Galactosidase