重组仿刺参超氧化物歧化酶在大肠杆菌中表达条件的优化及分离纯化
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摘要
利用基因工程菌BL21(DE3)/pET30a(+)-SOD1,优化表达重组仿刺参超氧化物歧化酶蛋白(rAj-SOD1)。采用摇瓶发酵优化rAj-SOD1可溶性表达的条件,提高蛋白的表达产量。结果表明,在温度30℃、转速200 r/min、0.1 mmol/L IPTG诱导8 h条件下,菌体中rAj-SOD1蛋白表达量最大,达到315.59 mg/L。电泳检测结果表明,rAj-SOD1的纯度为96.7%,符合后续酶活要求。纯化后rAj-SOD1质量浓度为98.73 mg/L,收率为31.28%。邻苯三酚法对rAj-SOD1蛋白进行活性分析,结果表明,纯化的rAj-SOD1蛋白具有明显的抗氧化活性。
The recombinant E. coli BL21(DE3)/pET30 a(+)-SOD1 were used to express Apostichopus japonicus superoxide dismutase(rAj-SOD1). The fermentation conditions were optimized for rAj-SOD1 expression. The results showed that the expression level of rAj-SOD1 could reach to the highest(315.59 mg/L) at 30 ℃, 200 r/min and 0.1 mmol/L IPTG induction for 8 h. The electrophoresis result showed that the purity of rAj-SOD1 was 96.7%, which could meet the requirements of enzyme activities. The mass concentration of purified rAj-SOD1 was 98.73 mg/L and the yield was 31.28%. The activity of rAj-SOD1 was analyzed by pyrogallol method, and the result showed that the purified rAj-SOD1 had obvious antioxidant activity.
The recombinant E. coli BL21(DE3)/pET30 a(+)-SOD1 were used to express Apostichopus japonicus superoxide dismutase(rAj-SOD1). The fermentation conditions were optimized for rAj-SOD1 expression. The results showed that the expression level of rAj-SOD1 could reach to the highest(315.59 mg/L) at 30 ℃, 200 r/min and 0.1 mmol/L IPTG induction for 8 h. The electrophoresis result showed that the purity of rAj-SOD1 was 96.7%, which could meet the requirements of enzyme activities. The mass concentration of purified rAj-SOD1 was 98.73 mg/L and the yield was 31.28%. The activity of rAj-SOD1 was analyzed by pyrogallol method, and the result showed that the purified rAj-SOD1 had obvious antioxidant activity.
引文
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[2] BORDBAR S, ANWAR F, SAARI N. High-value components and bioactives from sea cucumbers for functional foods: a review[J]. Marine Drugs, 2011, 9(10): 1761-1805.
[3] BRUCKNER A W, JOHNSON K A, FIELD J D. Conservation strategies for sea cucumbers: can a CITES Appendix Ⅱ listing promote sustainable international trade[J]. SPC Beche-de-mer Information Bulletin, 2003, 18: 24-33.
[4] KIEW P L, DON M M. Jewel of the seabed: sea cucumbers as nutritional and drug candidates[J]. International Journal of Food Sciences and Nutrition, 2012, 63(5): 616-636.
[5] YI J F, LI Y M, LIU T, et al. Mn SOD and CuZn SOD polymorphisms and interactions with risk factors in gastric cancer[J]. World Journal of Gastroenterology, 2010, 16(37): 4738-4746.
[6] JOHNSON F, GIULIVI C. Superoxide dismutases and their impact upon human health[J]. Molecular Aspects of Medicine, 2005, 26(4/5): 340-352.
[7] 顾景范,杜寿玢,查良锭,等.现代临床营养学[M].北京:科学出版社,2003.
[8] BARJA G, HERRERO A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals[J]. The FASEB Journal, 2000, 14(2): 312-318.
[9] CRUZ M E, MANUELA G M, BARBARA M, et al. Liposomal superoxide dismutases and their use in the treatment of experimental arthritis[J]. Methods in Enzymology, 2005, 391: 395-413.
[10] CHEN Y P, CHEN C T, HUNG Y, et al. A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: superoxide dismutase[J]. Journal of the American Chemical Society, 2013, 135(4): 1516-1523.
[11] SHARMA N C, SAHI S V, JAIN J C. Sesbania drummondii cell cultures: ICP-MS determination of the accumulation of Pb and Cu[J]. Microchemical Journal, 2005, 81(1): 163-169.
[12] LIU J, WANG J, YIN M, et al. Purification and characterization of superoxide dismutase from garlic[J]. Food and Bioproducts Processing, 2011, 89(4): 294-299.
[13] PARK K Y, KIM E Y, LEE W, et al. Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.)[J]. Protein Expression and Purification, 2016, 119: 69-74.
[14] BRADFORD M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1/2): 248-254.
[15] 韩少华,朱靖博,王妍妍,等.邻苯三酚自氧化法测定抗氧化活性的方法研究[J].中国酿造,2009,28(6):155-157.
[16] 陈颖,沈敏,刘宝林,等.包涵体蛋白纯化方法的探讨[J].放射免疫学杂志,2011,24(1):62-64.