摘要
目的:实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,并对表达产物进行活性测定。方法:用PCR方法从大肠杆菌2号基因组中扩增Mn-SOD基因编码区,克隆到pUCm-T Vector,测定核苷酸序列;再将基因编码区克隆到原核表达载体PET-28a,构建含Mn-SOD基因的重组表达质粒,转化到大肠杆菌BL21中进行IPTG诱导表达。结果:SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50%;黄嘌呤氧化酶法测定表达蛋白活性,菌体可溶性总蛋白中表达产物酶比活为3921.8 U/mg,是对照BL21的276.8倍。结论:Mn-SOD基因可在BL21中成功表达,产物具有高可溶性、高活性的特点。
Objective： To achieve the soluble expression of Mn-SOD gene in E.coli and assay the enzyme activity of the expressed product. Methods： The coding region of superoxide dismutase was amplified using PCR method from the E.coli genome. The PCR product was cloned into PUC19-T vector and sequenced.In addition, the cloned coding region of Mn-SOD was inserted into the expression vector PET-28a to form the recombinant plasmid PET-28a-Mn-SOD and was then transformed into E.coli BL21 for expression. Results： The SDS-PAGE analysis revealed that the expression recombinant SOD accumulated up to 50% of the total bacterial protein. The enzyme activity of Mn-SOD was assayed with xanthine oxidase method. The result showed that the specific activity of the expressed product in the total soluble protein was 3921.77 U/mg, and was 276.77 times of.E.coli BL21. Conclusion ： Mn-SOD gene can be success fully expressed in E.coli products hate the chardcteristics of high solubihity ard high activity.
出处
《温州医学院学报》
CAS
2009年第1期53-56,共4页
Journal of Wenzhou Medical College
