Cloning of cholesterol oxidase gene from Iranian strain of Rhodococcus for production of cotton biological pesticide

Cholesterol oxidase enzyme, as a new generation of biological pesticides, has pesticides strong effect on the pest to cotton Heliothis armigera. The mechanism of action of cholesterol oxidase fatality related to the oxidation of cholesterol in the membranes of insect midgut epithelial which leads to physical destruction and the resulting disturbances in membrane structure and function and eventually death of the insect. In order to retrofitting genetically engineered plants against pests, production of them with the gene coding for cholesterol oxidase enzyme, require the best method for expression level and activity of this enzyme. Therefore, this study aimed to clone cholesterol oxidase enzyme in plasmid pET23a for possible guidance recombinant protein produced in bacterial periplasmic space. Rhodococcus sp. strain 502 that produce high level of cholesterol oxidase, was used for gene isolation by PCR. The PCR product was sub cloned in plasmid pJET, subsequently for expression was cloned in pET23a, eventually was transformed into the host E.coli DH5α. Recombinant plasmid pET23a CHO was confirmed by restriction enzyme digestion and sequencing. The results of sequencing showed high similarity between our isolated gene sequence and the other cholesterol oxidase gene sequences in NCBI that can be used for production of biological pesticide.