DNA LIGASE-CODING GENE OF HYPERTHERMOPHILIC AQUIFEX PYROPHILUS AND PROTEIN EXPRESSED THEREFROM

摘要

PURPOSE: Provided is a DNA ligase-coding gene of hyperthermophilic Aquifex pyrophilus. And protein expressed therefrom is also provided, which is stable at high temperatures and useful for assay methods in medical industry. CONSTITUTION: The DNA ligase-coding gene of hyperthermophilic Aquifex pyrophilus is obtained by the next steps of: i) cultivating Aquifex pyrophilus to obtain DNA; ii) cutting DNA with restriction enzyme HindIII to obtain small segments; iii) inserting the segments into vector pBluescript KS(±) and obtaining clones having segments of DNA ligase through polymerase chain reaction; iv) separating DNA segments included in the clones, 400bp, on agarose gel and making genome library; v) selecting clones showing positive signal when put on ECL direct system; vi) obtaining DNA from the clones and cutting DNA with restriction enzyme HindIII; vii) performing Southern blotting with DNA segments; viii) obtaining segments showing positive signal and linking the segments with pBluescript KS(±); and ix) determining the base sequence of the clones obtained.