关键词: 牦牛, Cu, Zn-SOD, 克隆, 原核表达

Abstract:

【Objective】 Due to the medical superoxide dismutase (SOD) source is restricted from animals and plants, establishment of the prokaryotic expressing system of yak Cu, Zn-SODpET22b(+)-E. coli BI21(DE3) by genetic engineering method is a basis for recombinant SOD protein of clinical injection. 【Method】 Yak Cu, Zn-SOD gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was inserted into the vector pET22b(+) to construct plasmid Cu, Zn-SOD/ pET22b(+), then the plasmid expressed in E. coli BL21 (DE3) cell that induced by IPTG. The expression product and it's activity were tested by the method of SDS-PAGE, native PAGE (enzyme active stain) electrophoresis and pyrogallol (1,2,3-trihydroxybenzene) self oxidation. The level of expression protein was determined by Brad Ford. 【Result】 The length of ORF of yak Cu, Zn-SOD was 456 bp encoding 152 amino acids peptides. The nucleotide sequence and amino acids sequence of Cu, Zn-SOD similarities between yak and bovine were 99.6% and 98.7%, respectively. The activity ratio of the protein crude extract of recombinant Cu, Zn-SOD was 35U?mg-1. The concentration of the recombinant Cu, Zn-SOD is 0.2772 (mg?mL-1). 【Conclusion】 The prokaryotic expression vector for yak Cu, Zn-SOD/pET22b(+) was constructed successfully and it expresses in Escherichia coli stably and highly. The recombinant product was obtained and the product has higher biological activity.

Key words: yak, Cu, ZnSOD, clone, prokaryotic expression