溶藻弧菌PepA蛋白原核表达载体的构建及其乙酰化鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0444

摘要/Abstract

摘要：

旨为构建溶藻弧菌 HY9901 PepA蛋白的原核表达载体、优化其表达条件,并分析该蛋白是否存在乙酰化调控。首先设计特异性引物经PCR克隆 pepA基因,构建表达载体pET-28a-PepA并将其转入大肠杆菌BL21（DE3）,然后用SDS-PAGE和Western blot分析蛋白的表达及乙酰化情况。结果显示,表达菌株可以正确表达重组蛋白（60.7 kD）,其最佳表达条件为：体积分数为0.1%的IPTG,37℃诱导5 h;Western blot结果表明,PepA蛋白是乙酰化蛋白,且在体外不存在去乙酰化。

关键词: 溶藻弧菌, PepA蛋白, 表达条件优化, 乙酰化

Abstract:

The purpose of this study is to construct a prokaryotic expression vector of PepA protein in Vibrio alginolyticus HY9901,to optimize its expression conditions,and to analyze whether or not it is regulated by protein acetylation. Firstly,the pepA gene was cloned via PCR,and an expression vector pET-28a-PepA was constructed and transferred it into Escherichia coli BL21（DE3）.Then,the protein expression and acetylation were analyzed by SDS-PAGE and Western blot. The recombinant expression strain correctly expressed the recombinant protein（60.7 kD）,and its optimal expression conditions was,induced for 5 h at 37℃ by IPTG with a volume fraction of 0.1%. Western blot results showed that PepA protein was acetylated protein,and there was no deacetylation in vitro.

Key words: Vibrio alginolyticus, PepA protein, optimal expression condition, acetylation

徐洲, 范晨龙, 丁燏. 溶藻弧菌PepA蛋白原核表达载体的构建及其乙酰化鉴定[J]. 生物技术通报, 2020, 36(12): 75-81.

XU Zhou, FAN Chen-long, DING Yu. Construction of Prokaryotic Expression Vector of PepA Protein of Vibrio alginolyticus and Identification of Its Acetylation[J]. Biotechnology Bulletin, 2020, 36(12): 75-81.

图/表 8

图1 pepA基因的克隆 M：DL2000 DNA分子标准;1-4：pepA基因扩增结果

图2 构建pET-28a-PepA载体 M：DL2000 DNA分子标准;1-7：pET-28a-Pepa的PCR产物

图3 PepA蛋白SDS-PAGE电泳分析 M：26616蛋白质分子标准;1：pET-28a-PepA（诱导）;2：pET-28a-PepA（未诱导）;3：pET-28a（诱导）;4：pET-28a（未诱导）

图4 温度对PepA蛋白表达的影响 M：26616蛋白质分子标准;1：37℃下诱导的全菌蛋白;2-3：37℃下诱导的可溶性蛋白和包涵体蛋白;4：28℃下诱导的全菌蛋白;5-6：28℃下诱导的可溶性蛋白和包涵体蛋白

图5 IPTG浓度对PepA蛋白表达的影响 M：26616蛋白质分子标准;1-7：IPTG浓度分别为0.1%、0.2%、0.4%、0.6%、0.8%、1%和2%的条件下诱导

图6 诱导时间对PepA蛋白表达的影响 M：26616蛋白质分子标准;1-7：诱导时间为别为1 h,2 h、3 h、4 h、5 h、6 h和7 h的条件下诱导

图7 PepA蛋白的纯化 M：26616蛋白质分子标准;1-2：流穿液1-2;3-4：洗涤液1-2;5-9：洗脱液1-5

图8 PepA蛋白的乙酰化鉴定及调控 M：26616蛋白质分子标准;1：200 μL PBS*组;2：100 μL NAD+和100 μL PBS*组;3：100 μL CobB和100 μL PBS*组;4：100 μL NAD+和100 μL CobB组;5：E3蛋白

参考文献 28

[1]	Robert-Pillot A, Guenole A, Fournier JM. Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species:validation by DNA-DNA hybridization[J]. FEMS Microbiol Lett, 2002,215(1):1-6. doi: 10.1111/j.1574-6968.2002.tb11362.x URL pmid: 12393193
[2]	梅冰, 陆翔, 王丽娜, 等. 溶藻弧菌的毒力因子与相关基因的研究进展[J]. 辽宁农业科学, 2015,1(5):58-60.
	Mei B, Lu X, Wang LN, et al. Progress on virulence factor and virulence gene research of Vibrio alginolytic[J]. Liaoning Agricultural Sciences, 2015,1(5):58-60.
[3]	周泽军. 溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白VscO的功能研究[D]. 湛江:广东海洋大学, 2013.
	Zou Z. Characterization of the chaperone escort protein VscO involved in type III secretion system in marine fish pathogen Vibrio alginolyticus[D]. Zhanjiang:Guangdong Ocean University, 2013.
[4]	丁淑妍, 侯丽艳, 于成勇, 等. 海洋性弧菌致病机制研究进展[J]. 职业与健康, 2019,35(7):984-989.
	Ding SY, Hou LY, Yu CY, et al. Research progress on pathogenic mechanism of marine Vibrios[J]. Occupation and Health, 2019,35(7):984-989.
[5]	Austin B. Vibrios as causal agents of zoonoses[J]. Vet Microbiol, 2010,140(3-4):310-317.
[6]	Ewert J, Glück C, Strasdeit H, et al. Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii[J]. Enzyme and Microbial Technology, 2018,110(1):69-78.
[7]	Yin KY, Peng Y, Ahmed MAH, et al. PepA binds to and negatively regulates esrB to control virulence in the fish pathogen Edwardsiella piscicida[J]. Microbiol Res, 2020,232(1). doi: 10. 1016/j. micres. 2019. 126349.
[8]	Nguyen LMP, Nadal M, Charlier D. The trigger enzyme PepA(aminopeptidase A)of Escherichia coli, a transcriptional repressor that generates positive supercoiling[J]. FEBS Lett, 2016,590(12):25-1816.
[9]	杨燕, 罗林根, 徐妙, 夏立秋. 亮氨酰氨肽酶基因的阻断对刺糖多孢菌生长及次级代谢产物合成的影响[J]. 微生物学报, 2016,56(4):629-642.
	Yang Y, Luo L, Xu M, Xia LQ. Disruption of leucyl aminopeptidase gene affects phenotypes and second metabolite production of Saccharopolyspora spinosa[J]. Acta Microbiologica Sinica, 2016,56(4):629-642.
[10]	Reverdy A, Chen Y, Hunter E, et al. Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity[J]. PLoS One, 2018,13(9):e0204687. URL pmid: 30265683
[11]	徐雅琴, 付雪婷, 王丽波, 等. 乙酰化修饰对黑穗醋栗果实多糖结构特性及活性的影响[J]. 精细化工, 2019,36(12):2467-2475.
	Xu YQ Fu XT Wang LB, et al. Effects of acetylation modification on structural characteristics and bioactivities of polysaccharides from Black Currant[J]. Fine Chemicals, 2019,36(12):2467-2475.
[12]	Yang QZ, Liu XR, Chen J, et al. Lead-mediated inhibition of lysine acetylation and succinylation causes reproductive injury of the mouse testis during development[J]. Toxicol Lett, 2020,318(1):30-43.
[13]	冼钰茵, 余翀, 阮荣勇, 等. 广州市售水产品副溶血弧菌和溶藻弧菌的耐药性评估[J]. 安徽农业科学, 2017,45(28):74-77.
	Xian YY, Yu CY, Ruan RY, et al. Drug resistance of Vibrio parahemolytics and Vibrio alginolyticus in aquatic products from Guangzhou retail markets[J]. Journal of Anhui Agricultural Sciences, 2017,45(28):74-77.
[14]	马畅, 李婷, 刘士尧, 等. 蛋白乙酰化酶GNAT调控水稻细菌性条斑病菌的致病力[J]. 海南大学学报:自然科学版, 2019,37(1):29-33.
	Ma C, Li T, Liu SY, et al. GNAT Regulates the Virulence of Xanthomonas oryzae pv. oryzicola[J]. Natural Science Journal of Hainan University, 2019,37(1):29-33.
[15]	任洁. 乙酰化修饰调节PhoP活性以及鼠伤寒沙门菌毒力机制研究[D]. 上海:上海交通大学, 2017.
	Ren J. The Role of Acetylation in Regulating PhoP Activity and Salmonella Virulence[D]. Shanghai:Shanghai Jiao Tong University, 2017.
[16]	倪进婧, 姚玉峰. 细菌核糖体蛋白RpIB的乙酰化修饰及对其功能的影响[C]. 中华医学会第十二次全国临床微生物学术年会暨第十一次全球华人临床微生物学与感染症学术年会论文汇编, 2015: 154.
	Ni JJ, Yao YF. Acetylation modification of bacterial ribosomal protein RpIB and its effect on its function[C]. The Twelfth National Academic Conference on Clinical Microbiology and the Eleventh Global Chinese Academic Annual Conference on Clinical Microbiology and Infectious Diseases of the Chinese Medical Association, 2015: 154.
[17]	方鹏, 陈婷, 钟琦, 等. 组蛋白乙酰化在认知功能障碍中的研究进展[J]. 临床神经病学杂志, 2019,32(5):380-382.
	Fang P, Chen T, Zhong Q, et al. Research progress of histone acetylation in cognitive dysfunction[J]. Journal of Clinical Neurology, 2019,32(5):380-382.
[18]	赵敏, 陈垚, 李旭光, 等. 低氧环境下蛋白质乙酰化修饰研究学术进展与存在的问题[J]. 中国组织工程研究, 2019,23(34):5570-5576.
	Zhao M, Chen Y, Li XG, et al. Protein acetylation in hypoxic environment:research advance and the existing problems[J]. Chinese Journal of Tissue Engineering Research, 2019,23(34):5570-5576.
[19]	邹波, 李春保. 乙酰化、能量代谢与肉品品质的关联[J]. 肉类研究, 2019,33(8):58-64.
	Zou B, Li CB. Association of meat quality with acetylation and energy metabolism[J]. Meat Research, 2019,33(8):58-64.
[20]	Eldeeb Mohamed A, Fahlman Richard P, Ragheb Mohamed A, et al. Does N-terminal protein acetylation lead to protein degradation?[J]. Bioessays, 2019,41(11):e1800167. URL pmid: 31549739
[21]	Aksnes H, Ree R, Arnesen T. Co-translational, post-translational, and non-catalytic roles of N-terminal acetyltransferases[J]. Mol Cell, 2019,73(6):1097-1114. URL pmid: 30878283
[22]	杨雨薇, 尚爽, 胡卓伟, 等. 赖氨酸乙酰化的研究进展及应用[J]. 药学学报, 2019,54(5):778-787.
	Yang YW, Shang S, Hu ZW, et al. Advances and applications of lysine acetylation[J]. Acta Pharmaceutica Sinica, 2019,54(5):778-7873.
[23]	周泽军, 庞欢瑛, 丁燏, 等. 溶藻弧菌HY9901转运蛋白TolB的原核表达及条件优化和纯化[J]. 中国农学通报, 2013,29(11):55-59.
	Zou ZJ, Pang HY, Ding Y, et al. Purification and optimization of prokaryotic expression of translocation protein TolB gene from Vibrio alginolyticus strain HY9901[J]. Chinese Agricultural Science Bulletin, 2013,29(11):55-59.
[24]	陈立明, 庞欢瑛, 简纪常, 等. 溶藻弧菌dtd基因的克隆及原核表达条件优化[J]. 广东海洋大学学报, 2014,34(3):52-57.
	Chen LM, Pang HY, Jian JC, et al. Cloning and optimization of prokaryotic expression of D-Tyr-tRNA ^Tyr deacylase(DTD)gene from Vibrio alginolyticus [J]. Journal of Guangdong Ocean University, 2014,34(3):52-57.
[25]	丁军颖, 伊瑶, 卢学新, 等. 不同温度、时间、IPTG和菌种浓度对丁肝抗原蛋白表达量的影响[J]. 医学研究杂志, 2012,41(5):31-34.
	Ding JY, Yi Y, Lu XX, et al. Effect of different temperature, Time and different concentration of IPTG and strains on HDVAg expression[J]. Journal of Medical Research, 2012,41(5):31-34.
[26]	Christensen DG, Meyer JG, Baumgartner JT, et al. Identification of novel protein lysine acetyltransferases in Escherichia coli.[J]. mBio, 2018,9(5):e01905-18. URL pmid: 30352934
[27]	叶青, 王恩多. 原核生物中蛋白质的赖氨酸乙酰化修饰[J]. 生命科学, 2016,28(4):427-435.
	Ye Q, Wang ED. Lysine acetylation of proteins in prokaryotes[J]. Chinese Bulletin of Life Sciences, 2016,28(4):427-435.
[28]	Spange S, Wagner T, Heinzel T, et al. Acetylation of non-histone proteins modulates cellular signalling at multiple levels[J]. Int J Biochem Cell Biol, 2009. 41(1):98-185.

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