Use of conversion adaptors to clone antigen genes in lambda gt11.
A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.
D000942:Antigens, Bacterial / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D009838:Oligodeoxyribonucleotides; D012285:Orientia tsutsugamushi / Q000235:genetics / Q000276:immunology
D000942:Antigens, Bacterial; D009838:Oligodeoxyribonucleotides