Cloning Of Cholesterol Oxidase Gene From Rhodococcus Equi And Its Expression In Escherichia Coli

Cholesterol oxidase is a type of protein that was found to have pest-resistant activity only years ago, its toxity to such sensitive pests as cotton boll weevil is euqivalent to previously found Bt. endotoxins . The protein can also inhibit the growth and development of cotton boll worm. Because of its high toxity activity and complementary inseticidal spectrum with Bt. endotoxin which is now being used universally ,the cholesterol oxidase is acknowleged as one of the representitive of the insecticidal protein of the second generation . Moreover, together with cholesterol esterase and catalyse, cholesterol oxidase can be used as test reagent to determine the total cholesterol content in blood plasma for the diagnosis of such as cardiovascular reflex and atherosclerosis clinically. Therefore, research on cholesterol oxidase has become more and more important.A 1.6 kb DNA fragment was amplified using PCR method from genomic DNA of Rhodococcus equi. 4-2G2. Sequence analysis showed that, this fragment has a homology of 99% to the previously reported choE from Rhodococcus equi. after cloning and subcloning and three identical fragments were obtained from three independent PCR, lacking three continual bases (TTC, encoding the Pheamino acid) compared with choE, thus it can be assumed that the new fragment, designated as choEW, and choE belong to the same gene family, even it might be the natural mutation of choE. Different expression systems were constructed using different expression vector including pET-His and E. coli TOPI OF' and BL21 host cells. The expression mixture were eletrophoresized by SDS-PAGE, revealing a specific 56 kD protein in BL21 expression system, which is of the same size in expectation. A colorimetric assay for measuring cholesterol oxidase showed the cholesterol oxidase activity of the expression product.In this study, a new gene c/wEW, encoding cholesterol oxidase, was isolated from Rhodococcus equi. 4-2G2 found in China, which may be useful in clinical diagnosis healthy food and pest management in agriculture. In addition, the gene has been expressed successfully, the expression product has cholesterol oxidase activity, thus this work provided theoretical basis to the development of genetic engineering of cholesterol oxidase.