人铜锌超氧化物歧化酶基因改良及在聚球藻中表达

Sited-directed mutagenesis of hCu,Zn-SOD gene and its expression in Synechococcus sp.PCC7942

应用PCR定点突变技术把质粒pESOD中人铜锌超氧化物歧化酶基因(hCu,Zn-SOD)的Cys111密码子突变为Ala111密码子,再构建重组子,通过随机同源重组将突变后的hCu,Zn-SOD整合入聚球藻Synechococcussp.PCC7942,并实现表达。表达产物用SDS-PAGE、Western blot、酶活等方法测定均为阳性反应;热稳定性测定显示,hCu,Zn-SOD在80℃保温30min后仍具有95%的活力,耐热能力比天然hCu,Zn-SOD有了较大的提高。蛋白扫描结果显示目的蛋白占可溶性蛋白的3.61%。

The Cys^111 genetic code of human copper/zinc superoxide dismutase （hCu, Zn-SOD ） gene in the pESOD plamid was mutated into the Ala^111 code with site-directed mutagenesis, and then the plamid pESODT^111 which contained groESL promoter, mutated hCu, Zn-SOD gene, rbcS-polyA terminator and reporter gene（ Kan^r） was constructed and transduced into Synechococcus sp. PCC7942 with homologous recombination platform. The results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host ceil. SDS-PAGE,Western blot and Pyrogallol autoxidation assay confirmed that the transformant strains expressed the mutated hCu,Zn-SOD protein. And the level of the mutated hCu,Zn-SOD protein reached a value of 3.61 % of the total soluble protein. Furthermore, the transformants still retained 95% activities of SOD after 30 minutes at 80℃ environment, it indicated that the mutated hCu, Zn-SOD protein could endure higher temperature than the natural one.