溶藻弧菌去乙酰化酶基因cobB克隆及其功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-1361

摘要/Abstract

摘要：

旨在研究溶藻弧菌（Vibrio alginolyticus）去乙酰化酶cobB基因的功能。进行了cobB基因的克隆,生物信息学分析,CobB蛋白质诱导表达、纯化及功能研究。CobB蛋白质的相对分子质量为27.09750 kD,理论等电点为5.15,化学式为C₁₁₈₆H₁₈₆₅N₃₃₉O₃₆₉S₁₀,亲水性平均系数（GRAVY）：-0.409,为亲水蛋白。CobB与同弧菌属的魔鬼弧菌（Vibrio diabolicus）,哈维弧菌（Vibrio harveyi）,霍乱弧菌（Vibrio cholerae）和副溶血性弧菌（Vibrio parahaemolyticus）具有高度同源性,在革兰氏阴性菌中相对保守;融合蛋白大小约为53 kD,去除标签后CobB蛋白分子量约为27 kD;CobB蛋白最适表达条件为37℃,0.4 mmol /L IPTG下诱导4 h。功能分析表明,CobB对乙酰化蛋白具有去乙酰化作用。

关键词: 溶藻弧菌, CobB, 去乙酰化, 蛋白表达

Abstract:

This work aims to study the function of the histone deacetylase cobB gene in Vibrio alginolyticus. The clone of cobB gene,its bioinformatics analysis,and induction,expression,purification and function of CobB protein were carried out. The relative molecular mass was 27.09750 kD,theoretical isoelectric point was 5.15,the chemical formula of the protein was C₁₁₈₆H₁₈₆₅N₃₃₉O₃₆₉S₁₀,and hydrophilic average coefficient grand aural of hydropathity（GRAVY）：-0.409,showing that it was a hydrophilic protein. CobB presented high homology with that of Vibrio diabolicus,Vibrio harveyi,Vibrio cholerae,and Vibrio parahaemolyticus,and was relatively conserved among Gram-negative bacteria. The fusion protein was about 53 kD,and the molecular weight of the CobB protein after tag removed was about 27 kD. The optimal expression condition of the CobB protein was 37℃ and inducing time 4 h with 0.4 mmol/L IPTG. Functional analysis showed that the CobB had deacetylation effect on acetylated protein.

Key words: Vibrio alginolyticus, CobB, deacetylation, protein expression

范晨龙, 丁燏. 溶藻弧菌去乙酰化酶基因cobB克隆及其功能验证[J]. 生物技术通报, 2021, 37(8): 195-202.

FAN Chen-long, DING Yu. Molecular Cloning and Functional Verification of Histone Deacetylase Gene cobB in Vibrio alginolyticus[J]. Biotechnology Bulletin, 2021, 37(8): 195-202.

图/表 10

图1 cobB基因全长PCR克隆结果 M：DNA marker;1-4：目的基因 PCR 扩增

Fig.1 Results of cobB full-length PCR cloning M：DNA marker;1-4：PCR amplified product of target gene

图2 cobB 序列生物信息学分析 A：信号肽预测;B：跨膜结构域预测;C：蛋白质三级结构预测

Fig.2 Bioinformatics analysis of cobB sequences A：Signal peptide prediction. B：Transmembrane domain prediction.C：Protein tertiary structure prediction

图3 CobB氨基酸序列系统发育树

Fig.3 Phylogenetic tree based on CobB sequences

图4 CobB蛋白表达观测实验凝胶电泳示意图 M：蛋白质 marker;1：pGEX-6P-1未诱导;2：pGEX-6P-1诱导;3：pGEX-6P-1-cobB 未诱导;4：pGEX-6P-1-cobB 诱导

Fig.4 Schematic diagram of gel electrophoresis of CobB protein expression M：Protein marker;1：negative control without IPTG;2：negative control with IPTG;3：pGEX-6p-cobB without IPTG;4：pGEX-6p-cobB with IPTG

图5 不同诱导时间对 CobB 蛋白表达影响凝胶电泳示意图 M：蛋白质 marker;1-7：诱导时间分别为1 h、2 h、3 h、4 h、5 h、6 h、7 h

Fig.5 Schematic diagram of gel electrophoresis of different induction time on CobB protein expression M：Protein marker;1-7：The induction time is 1 h、2 h、3 h、4 h、5 h、6 h、7 h

图6 不同IPTG浓度对 CobB 蛋白表达影响凝胶电泳示意图 M：蛋白质 marker;1-7：IPTG浓度分别为 0.1%、0.2%、0.4%、0.6%、0.8%、1% 和 2%

Fig.6 Schematic diagram of gel electrophoresis of different IPTG concentrations on CobB protein expression M：Protein marker;1-7：the IPTG concentrations are 0.1%、0.2%、0.4%、0.6%、0.8%、1% and 2%

图7 不同温度对 CobB 蛋白表达影响凝胶电泳示意图 M：蛋白质marker;1：28℃全菌蛋白;2：28℃破碎后上清;3：28℃破碎后沉淀;4：37℃全菌蛋白;5：37℃破碎后上清;6：37℃破碎后沉淀

Fig.7 Schematic diagram of gel electrophoresis of different temperatures on CobB protein expression M：Protein maker;1：28℃ bacteria protein;2：28℃ supernatant protein;3：28℃ precipitation protein;4：37℃ bacteria protein;5：37℃ supernatant protein;6：37℃ precipitation protein

图8 CobB 蛋白纯化酶切凝胶电泳示意图 M：蛋白质 Marker;1：全菌蛋白;2：CobB融合蛋白纯化结果;3：CobB融合蛋白酶切结果

Fig.8 Schematic diagram of digestion gel electrophoresis of CobB protein purification M：Protein molecular standard;1：Bacteria protein;2：Results of CobB fusion protein purification;3：Results of CobB fusion protease cleavage results

图9 CobB 去乙酰化 GlmU 蛋白的 Western 印迹 A：乙酰赖氨酸特异性抗体的 Western 印迹及 SDS-PAGE;B：Western 印迹定量的 GlmU 乙酰化水平。1：无CobB蛋白,无NAD +组;2：有CobB蛋白,无NAD+组;3：无CobB蛋白,有NAD +组;4：有CobB蛋白,有NAD +组

Fig.9 Western blot of CobB deacetylated GlmU protein A：Western blot of acetyllysine-specific antibodies and SDS-PAGE;B：GlmU acetylation level quantified by Western blot. 1：No CobB protein,no NAD+ group;2：CobB protein,no NAD + group;3：no CobB protein,NAD+ group;4：CobB protein,NAD+ group

图10 CobB融合蛋白及去除标签的CobB蛋白去乙酰化GlmU蛋白的Western 印迹 A：乙酰赖氨酸特异性抗体的Western印迹及SDS-PAGE;B：Western印迹定量的GlmU乙酰化水平。1：CobB 融合蛋白组;2：CobB蛋白组;3.GST蛋白组

Fig.10 Western blot of CobB fusion protein and CobB protein deacetylated GlmU protein A：Western blot of acetyllysine-specific antibodies and SDS-PAGE;B：GlmU acetylation level quantified by Western blot. 1：CobB fusion protein group;2：CobB protein group;3：GST protein group

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