Expression of the Haemophilus parasuis sodA gene and the study on its physicochemical properties

doi:10.14088/j.cnki.issn0439-8114.2021.10.029

Abstract

Abstract: In order to study the activity and physiological function of the SOD protein encoded by the sodA gene of Haemophilus parasuis, the sodA gene was amplified from HPS by the specific primers and expressed in Escherichia coli prokaryotic expression system. The expressed protein was purified by the ammonium sulfate fractionation, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose Fast Flow anion exchange chromatography. The activity of the purified protein was determined by the pyrogallol autoxidation method and the physicochemical properties were studied by HPSEC and AAS. The results showed that the recombinant HPS SOD protein was a soluble protein with a size of 26 ku. The purity of the target protein was 95% after purification, and its activity was measured to be 215.9 U/mg by pyrogallol autoxidation assay. HPSEC analysis indicated that the recombinant HPS SOD was a multimer, and the manganese content in the purified HPS SOD was detected to be 0.71 mg/L by Flame Atomic Absorption Spectrophotometry.

Key words: Haemophilus parasuis, superoxide dismutase, pyrogallol autoxidation method

CLC Number:

S852.61

HU Ji-xiong, WANG Xi, LI Guo-pan, RONG Jun. Expression of the Haemophilus parasuis sodA gene and the study on its physicochemical properties[J]. HUBEI AGRICULTURAL SCIENCES, 2021, 60(10): 142-147.

References

[1] 迟阳. 副猪嗜血杆菌的分离鉴定及其16SrRNA和sodA基因的序列分析[D]. 长春:吉林农业大学, 2013.
[2] 程迪, 迟阳, 艾春华, 等. 副猪嗜血杆菌LN/111013株sod A基因序列分析及其毒力强度初步判断[J]. 中国兽医学报, 2015, 35(8): 1223-1227.
[3] BANNISTER W H.From haemocuprein to copper-zinc superoxide dismutase:A history on the fiftieth anniversary of the discovery of haemocuprein and the twentieth anniversary of the discovery of superoxide dismutase[J]. Free radical research, 1988, 5(1): 35-42.
[4] SEIB K L, WU H J, KIDD S P, et al.Defenses against oxidative stress in Neisseria gonorrhoeae:A system tailored for a challenging environment[J]. Microbiology and molecular biology reviews, 2006, 70(2): 344-361.
[5] KIM E J, CHUNG H J, SUH B, et al.Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor Müller[J]. Molecular microbiology, 1998, 27(1): 187-195.
[6] MILLER A F.Superoxide dismutases:Ancient enzymes and new insights[J]. Febs letters, 2012, 586(5): 585-595.
[7] SAN M L R, HOBBS M M, KAWULA T H. Periplasmic copper-zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide[J]. Molecular microbiology, 1998, 27(2):391-404.
[8] YESILKAYA H, KADIOGLU A, GINGLES N, et al.Role of manganese-containing superoxide dismutase in oxidative stress and virulence of Streptococcus pneumoniae[J]. Infection and immunity, 2000, 68(5): 2819-2826.
[9] NAJMULDEEN H, ALGHAMDI R, ALGHOFAILI F, et al.Functional assessment of microbial superoxide dismutase isozymes suggests a differential role for each isozyme[J]. Free radical biology and medicine, 2019, 134: 215-228.
[10] MACEDO N,ROVIRA A,TORREMORELL M.Haemophilus parasuis:Infection,immunity and enrofloxacin[J]. Veterinary research,2015, 46(1):1-6.
[11] GUO L, XU L, WU T, et al.Evaluation of recombinant protein superoxide dismutase of Haemophilus parasuis strain SH0165 as vaccine candidate in a mouse model[J]. Canadian journal of microbiology, 2017, 63(4): 312-320.
[12] 张昆丽, 李春玲. 副猪嗜血杆菌病研究进展[J]. 广东农业科学, 2020, 47(12): 166-174.
[13] 杨宇虹, 严洁, 李杰. 金黄色葡萄球菌锰超氧化物歧化酶的表达纯化及活性分析[J]. 中国病原生物学杂志, 2020, 15(1): 5-9.
[14] 张建康, 汪洋, 易力, 等. 鼠伤寒沙门菌超氧化物歧化酶SodA基因的表达及其酶活性的测定[J]. 中国兽医科学, 2017, 47(8): 975-980.
[15] 赵逸群. 荧光假单胞菌超氧化物歧化酶基因的克隆、表达及性质研究[D]. 山东青岛:青岛大学, 2018.
[16] HE L, HE T, FARRAR S, et al.Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species[J]. Cellular physiology and biochemistry, 2017, 44(2): 532-553.
[17] LU X, WANG C, LIU B.The role of Cu/Zn-SOD and Mn-SOD in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix[J]. Fish & shellfish immunology, 2015, 42(1): 58-65.
[18] XIKERANMU Z, MA J, LIU X.Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells[J]. Peer J, 2020, 8: e8507.
[19] 夏民旋, 王维, 袁瑞, 等. 超氧化物歧化酶与植物抗逆性[J]. 分子植物育种, 2015, 13(11): 2633-2646.
[20] BANEYX F.Recombinant protein expression in Escherichia coli[J]. Current opinion in biotechnology, 1999, 10(5): 411-421.
[21] SHI G, SHEN J, REN F, et al.Molecular cloning, expression, and characterization of BmSOD3 in silkworm (Bombyx mori)[J]. Archives of insect biochemistry and physiology, 2021, 106(1): e21744.
[22] 陈梦瑶, 冯超越, 南天豪, 等. 黄瓜Cu/Zn-SOD基因克隆及可溶性表达和纯化[J]. 生物技术, 2019, 29(2): 111-115.
[23] KHANI M H, BAGHERI M.Skimmed milk as an alternative for IPTG in induction of recombinant protein expression[J]. Protein expression and purification, 2020, 170: 105593.
[24] 秦松, 何雨峰, 况嘉铀, 等. Mn-SOD的提取及其应用研究进展[J]. 食品工业科技, 2019, 40(15): 363-367.
[25] 侯玉艳, 吴素蕊, 张丽, 等. 黑脉羊肚菌SOD的纯化及特性研究[J]. 食品工业科技, 2015, 36(15): 147-151.
[26] 任美凤, 胡瑞斌, 孙芳, 等. 鸡心SOD的分离纯化及部分性质研究[J]. 西南大学学报(自然科学版), 2013, 35(2): 35-40.
[27] 陈振雨. 探讨火焰原子吸收法测定水中锌的最佳条件[J]. 节能与环保, 2020(12): 54-55.
[28] 肖林, 程雅琴. 检测人血白蛋白中铝含量的石墨炉原子吸收分光光度法的建立[J]. 中国生物制品学杂志, 2001, 14(3): 178-180.
[29] 林庆斌, 廖升荣, 熊亚红, 等. 超氧化物歧化酶(SOD)的研究和应用进展[J]. 化学世界, 2006, 47(6): 378-381.
[30] 武金霞, 肖炜. 双孢蘑菇子实体超氧化物歧化酶(SOD)的分离纯化及部分性质[J]. 河北大学学报(自然科学版), 2002, 22(3): 264-267.
[31] EKANAYAKE P M, KANG H S, DE ZYOSA M, et al.Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone(Haliotis discus discus)[J]. Comparative biochemistry and physiology part B:Biochemistry and molecular Biology, 2006, 145(3-4): 318-324.
[32] 于小焱, 罗嫣, 张晓慧. 高效凝胶排阻色谱法测定牛血清白蛋白相对分子质量及其分布[J]. 化学分析计量, 2013, 22(5): 20-23,26.
[33] 王彦伟, 张素玲, 吴芃, 等. 鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定[J]. 中国畜牧兽医, 2020, 47(6): 1677-1684.