Use of Conversion Adaptors to Clone Antigen Genes in Lambda gt11

摘要

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning cohesive-ended restriction fragments into the unique EcoRI site of the gamma gt11 expression vector. Five gamma gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different cohesive ends. Recombinant phage 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 gamma gt11 expression libraries. Recombinant antigen expression was dependent on gamma ft11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic gamma gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides. Reprints. (jes)