Cloning Of Copper-Zinc Superoxide Dismutase Gene And Construction Of Recombined Vector In Lactobacillus Casei

Superoxide dismutase(SOD)is a metal-containing enzyme which can be found abundantly in microorganisms,plants or animals.Four types of SOD exist according to the present of metal cofactor at the enzyme active site:CuZn-SOD,Fe-SOD,Mn-SOD and Ni-SOD.They catalyse the dismutation of superoxide anion radical into molecular oxygen and hydrogen peroxide and related to the functions for against oxygen poison, resistance to radiation damage and anti-aging,and showed cure effects in some tumors, inflammatory diseases and self-immunity diseases.CuZn-SOD has been shown to be effective for treating inflammation,reducing reperfusion injuries,and decreasing blood pressure in animal model systems.Unfortunately,this enzyme is mainly separated and purificated from organism in nature,and its therapeutic potential is restricted because of its limited amount.Bioengineering techniques may solve this problem.However, currently,only few researches have been reported related to CuZn-SOD gene(sodC) cloning and expression.Lactobacillus casei is GRAS bacteria and has been used in food and pharmseutical industries extensively.However,this bacterium lacks of SOD and cannot be cultured in aerobic system,and its applications are limited in industry.If an SOD gene is transferred into this bacterium,its oxygen stress capacity may be improved.Therefore,the objectives of this research were to clone sodC from E.coli, to construct a sodC vector and to transfered it into the Lactobacillus casei.A series of experiments were conducted.In the first part of this study,a pair of primers,5'-GTG GAGCTC ACGGAGGTCCTATGAAACG-3' and 5'-CAG GGTAC C TACAGCGGATTTGCGACA-3',was designed to amplify sodC gene by PCR according to the published sequence of E.coli sodC gene with the software of Primer Premier 5.0.The PCR product was electrophorised and identified as a 522bp fragment. The sodC gene was cloned into the vector pMD18-T easy and the recombinant was named as pMD18-T-sodC and then transferred into the E.coli competent DH5a. Identifications with restriction enzymes,PCR and sequencing indicated that the sodC gene had been cloned successfully.In the second of this study,sodC gene was further inserted into XbaⅠand HindⅢmultiple cloning sites of the expression vector pMG36e.The positive recombinant was transferred into E.coli DH5a.Identified with corresponding restriction endonuclease XbaⅠand HindⅢand PCR method showed that the sodC gene had been recombined with pMG36e as designated and named as pMG36e-sodC.This recombinant vector was successfully transferred into Lactobaicllus casei by electrotransformation under 2.5kV,100Ωand 25μF.This research has established a high-technique platform and will create a brand-new avenue for expressing various functional genes in Lactobacillus.