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Use of conversion adaptors to clone antigen genes in λgt11

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
163
Issue
2
Identifiers
DOI: 10.1016/0003-2697(87)90241-7
Keywords
  • Recombinant Dna
  • Restriction Fragments
  • Antigens
  • Expression Vector
  • Rickettsia Tsutsugamushi
  • λGt11 Adaptors
Disciplines
  • Biology

Abstract

Abstract 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 λgt11 expression vector. Five λgt11 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 λgt11 expression libraries. Recombinant antigen expression was dependent on λgt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic λgt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.

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