PRODUCTION OF TERMINAL DEOXYNUCLEOTYDIL TRANSFERASE

摘要

PURPOSE:To efficiently obtain the subject enzyme for reagents for generic engineering by culturing an insect cell transformed by containing DNA coding a terminal deoxynucleotidyl transferase. CONSTITUTION:Whole DNA is prepared from a bovine thymus cDNAlambda phage library and protenase K is added to the whole DNA to incubate the whole DNA and the DNA is treated with phenol and precipitated by ethanol to afford a DNA and the DNA is subjected to PCR reaction using a sequence of formula I in the upstream of initiation codon of terminal deoxynucletidyltransferase(TdT) gene and a sequence of formula II in the downstream of terminating codon as primers to provide TdT gene. Then the TdT gene is integrated into Baculovirus vector to introduce the gene into insect cell and the transformed cell is cultured to manifest the gene and the product is subjected to anion exchange treatment and purified by hydroxyapatite chromatography to inexpensively and stably provide the objective TdT.