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

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