Use of conversion adaptors to clone antigen genes in lambda gt11

Area of Science:

• Molecular Biology
• Genomics
• Recombinant DNA Technology

Background:

• Lambda gt11 expression vectors are crucial for expressing foreign genes.
• Cloning 5' cohesive-ended restriction fragments into lambda gt11 can be challenging.
• Rickettsia tsutsugamushi antigens are important targets for diagnostics and therapeutics.

Purpose of the Study:

• To develop an efficient strategy for constructing lambda gt11 expression libraries.
• To clone 5' cohesive-ended restriction fragments of Rickettsia tsutsugamushi genomic DNA.
• To identify Rickettsia tsutsugamushi antigen-encoding sequences.

Main Methods:

• Employing EcoRI conversion adaptors for cloning into the lambda gt11 EcoRI site.
• Constructing five lambda gt11 chromosomal libraries using Rickettsia tsutsugamushi genomic DNA.
• Utilizing restriction enzymes to generate different 5' cohesive ends.

Main Results:

• Achieved high recombinant phage yields (up to 10(7) PFU) without library amplification.
• Successfully obtained sequences encoding all eight identified R. tsutsugamushi polypeptide antigens.
• Demonstrated that recombinant antigen expression occurred in 39% of recombinants upon lac promoter induction.

Conclusions:

• The devised strategy significantly enhances the efficiency of genomic lambda gt11 library construction.
• The method simplifies the removal of unligated oligonucleotides and eliminates blunt-ended ligation.
• This approach facilitates the identification of immunoreactive gene fragments from Rickettsia tsutsugamushi.