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维氏气单胞菌(Aeromonas veronii)是一种革兰氏阴性兼性厌氧菌,隶属于气单胞菌科、气单胞菌属[1]。有研究表明,这种新兴的水生病原菌可以引起人类和动物(包括水生动物)的腹泻、伤口感染和出血性败血症[2-3]。它兼具肠毒素、Ⅲ型分泌效应蛋白AexU、组氨酸激酶BvgS、丝氨酸蛋白酶、外膜蛋白和鞭毛等多种毒力因子[4-5],能给水产养殖业造成巨大的经济损失,并对食品安全造成威胁[6-7]。例如:养殖场的鲶鱼和黄颡鱼感染维氏气单胞菌后,会大量死亡[8-9]。因此,维氏气单胞菌引起的鱼类细菌性疾病是制约水产养殖发展的重要因素,而解构维氏气单胞菌的抗逆性对维氏气单胞菌的疾病防治具有重要意义。类CysB蛋白Cbl(CysB like protein)是LysR家族的转录调控因子。1995年,首次在大肠杆菌中发现了cbl基因,它与cys调控子的转录调控因子CysB蛋白非常相似,与CysB具有40%的氨基酸序列同源性[10]。在硫代谢中,Cbl作为细胞内硫酸盐水平的传感器,在体内和体外激活tau和ssu启动子,控制牛磺酸和脂肪族磺酸盐的转运及脱硫所需的tauABCD和ssuEADCB的转录,从而稳定细菌体内的硫酸盐水平[11-13]。Cbl的功能与CysB高度同一,目前对Cbl的研究较少,但鉴于CysB现有的功能,可以推断Cbl存在的潜在毒力控制、参与细菌体内氧化应激机制等重要功能[14-16]。因此,本研究以维氏气单胞菌为研究对象,对Cbl蛋白进行原核表达和纯化,旨在为进一步揭示Cbl蛋白的功能和机制奠定基础。
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维氏气单胞菌(Aeromonas veronii)C4菌株、大肠杆菌BL21(DE3)以及含pET-28a质粒的大肠杆菌DH5α均为课题组保存菌株。
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Bacteria DNA Extraction Kit、FastPure Gel DNA Extraction Mini Kit、FastPure Plasmid Mini Kit、2 × Phanta Max Master Mix以及2 × Rapid Taq Master Mix均购自Vazyme公司;限制性内切酶(EcoRI-HF和HindIII-HF)及Fast T4 DNA Ligase /Buffers均购自NEB公司;卡那霉素(Kan,水溶,储存的质量浓度为50 g·L−1,工作质量浓度为50 mg·L−1,−20 ℃保存)、异丙基−β−D 硫代半乳糖苷(IPTG,水溶,储存浓度为0.1 mol·L−1,工作浓度为0.1 mmol·L−1,−20 ℃保存)、聚丙烯酰胺混合液(29∶1,体积比)、Tris、乙二胺四乙酸(EDTA)、十二烷基硫酸钠(SDS)、过硫酸铵(APS)、四甲基乙二胺(TEMED)以及LB培养基(w=1%NaCl、w=1%蛋白胨和w=0.5%酵母提取物)均购自Solarbio公司。
Eppendorf Centrifuge 5418高速台式离心机(Eppendorf 中国有限公司)、Life ECO-PCR 基因扩增仪(杭州博日科技有限公司)、Bio-Rad MicroPulser电穿孔仪(美国Bio-Rad 公司)、HZQ-F100 振荡培养箱(哈尔滨市东联生化仪器有限公司)、SYNERGY-H1多功能酶标仪(美国BioTek 公司)、PowerPac™电泳仪(美国Bio-rad 公司)以及Typhoon FLA 9500多功能生物分子成像仪(浙江德力西电器有限公司)。
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设计cbl基因扩增引物(表1)。使用Bacteria DNA Extraction Kit提取维氏气单胞菌C4总DNA,以维氏气单胞菌C4基因组DNA为模板,利用cbl-F和cbl-R对cbl基因片段进行PCR扩增,其片段大小为948 bp。PCR反应体系为2 × Phanta Max Master Mix 25 μL,cbl-F(10 μmol·L−1)1 μL,cbl-R(10 μmol·L−1)1 μL,基因组DNA 1 μL,ddH2O 22 μL,反应总体积为50 μL。反应程序:94 ℃预变性10 min,94 oC变性30 s,55 ℃退火30 s,72 ℃延伸1 min,共30个循环,72 ℃后延伸10 min。PCR完成后进行琼脂糖凝胶(w=0.5%)电泳鉴定,再用FastPure Gel DNA Extraction Mini Kit纯化回收扩增的DNA片段。
表 1 引物序列
Table 1. Primer sequence
引物 Primers 核苷酸序列(5′→3′) Nucleotide sequence (5′→3′) 长度/bp Length cbl-F CCGGAATTCGTGAATTTTCAGCAGTTACG 29 cbl-R CCCAAGCTTTCAAATCTGGAAGTCGAG 27 -
使用FastPure Plasmid Mini Kit提取pET-28a质粒,再用EcoRI-HF和HindⅢ-HF对纯化回收的DNA片段和pET-28a质粒进行双酶切,酶切产物使用FastPure Gel DNA Extraction Mini Kit纯化回收后按一定比例(质粒∶片段的摩尔比为1∶5)使用Fast T4 DNA Ligase过夜连接,将扩增出的cbl基因连入pET-28a载体的EcoRI(GAATTC)~ HindⅢ(AAGCTT)位点之间。将连接产物通过电击转入大肠杆菌BL21(DE3)感受态细胞中,复苏后涂布于含卡那霉素LB平板,过夜培养后挑菌验证,阳性克隆子送往生工生物工程(上海)股份有限公司测序。
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将含有pET-28a-Cbl重组载体的大肠杆菌BL21(DE3)菌株接种至含有50 mg·L−1卡那霉素的LB液体培养基中,在37 ℃摇床培养至OD600在0.4~0.6之间,加入IPTG至终浓度为100 μmol·L−1,在150 r·min−1、15 ℃条件下,过夜诱导表达12 h。然后将菌液冷冻离心(4 ℃,4 000 r·min−1)10 min,用10 mL的 Tris-HCl(50 mmol·L−1 Tris,350 mmol·L−1 NaCl)缓冲液重悬菌体。超声破碎仪设置:超声时间5 s,间隙时间7 s,总时间25 min,温度25~28 ℃,功率35%。冰上超声破碎重悬的菌体后将破碎的菌体冷冻离心(4 ℃,12 000 r·min−1)10~15 min,分离得到沉淀和上清。提前取出镍柱,用20 mL Tris-HCl缓冲液清洗2~3遍后关闭镍柱阀门,将破碎上清注入镍柱中并封口,置于冰上150 r·min−1孵育1 h。打开镍柱阀门,流尽孵育上清,用50 mmol·L−1咪唑洗脱杂蛋白,100 mmol·L−1咪唑收集目的Cbl蛋白,500 mmol·L−1咪唑清洗镍柱。最后进行SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。
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剪下合适大小的透析袋,放入含有1 mmol·L−1 EDTA-2Na的w=2% NaHCO3溶液中煮10 min,去离子水清洗后放入1 mmol·L−1 EDTA-2Na溶液中再次煮10 min,再次用去离子水清洗透析袋。将诱导表达的蛋白加入透析袋中,用含有φ=30%甘油的Tris-HCl作为透析液于4 ℃进行透析,每6 h更换1次透析液,共透析12 h。取出透析袋中透析好的蛋白,SDS-PAGE观察目的条带,用BOSTER的BCA蛋白浓度测定试剂盒测量Cbl蛋白质量浓度。
Prokaryotic Expression and Purification of Cbl Protein from Aeromonas veronii
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摘要: 维氏气单胞菌(Aeromonas veronii)引起的细菌性疾病已威胁到鱼类养殖业的发展。Cbl(类CysB蛋白)是细菌硫代谢的重要调控因子之一。本研究选取pET-28a为Cbl的原核表达载体,以大肠杆菌BL21(DE3)为宿主菌,使用IPTG(Isopropyl-beta-D-thiogalactoside,异丙基−β−D−硫代半乳糖苷)诱导表达,用SDS-PAGE凝胶电泳验证蛋白条带,再用BOSTER的BCA蛋白浓度测定试剂盒测量Cbl蛋白浓度,寻找较适宜的蛋白表达条件。结果表明,当诱导时间为8 h,IPTG终浓度为0.1 mmol·L−1,诱导温度为15 ℃,咪唑洗脱浓度为100 mmol·L−1时,Cbl蛋白质量浓度能达到601.405 mg·L−1,可满足后续实验要求。Abstract: Bacterial diseases caused by Aeromonas veronii have threatened the development of fish production. And Cbl (CysB like protein) is one of the important regulatory factors of bacterial sulfur metabolism. Escherichia coli BL21 (DE3) was selected as the prokaryotic expression vector for Cbl, and culturedadded with IPTG (Isopropyl-beta-D-thiogalactoside) for culture to induce the expression of Cb1. The protein samples were run on SDS-PAGE gel electrophoresis to verify the protein bands of Cb1, and then the concentration of Cbl was determined with Boster’s BCA protein assay kit. The results showed that Cbl was successfully purified, and that the concentration of Cbl was up to 601.405 mg·L−1, which meets the requirements for subsequent experiments, when the induction time was 8h, the final IPTG concentration was 0.1 mmol·L−1, the induction temperature was 15 ℃ and the Imidazole concentration in the elution buffer was 100 mmol·L−1.
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Key words:
- Cbl /
- vector construction /
- prokaryotic expression /
- protein purification
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图 2 不同条件下Cbl蛋白的表达图谱
M:低分子质量蛋白Marker(14.4~97.4 KDa);1:诱导后培养2 h的沉淀;2:诱导培养后2 h的上清;3:诱导后培养4 h的沉淀;4:诱导后培养4 h的上清;5:诱导后培养6 h的沉淀;6:诱导后培养6 h的上清;7:空载pET-28a诱导后培养2 h的沉淀;8:未加IPTG诱导培养2 h的沉淀。
Fig. 2 Expression profile of Cbl protein under different conditions
M: Protein Marker (14.4kD-97.4 kDa); 1: The precipitation of the induced sample cultured for 2 h; 2: The supernatant of the sample cultured for induction for 2 h; 3: The precipitation of the sample cultured for induction for 4 h; 4: The supernatant of the sample cultured for induction for 4 h; 5: The precipitation of the sample cultured for induction for 6 h; 6: The supernatant of the sample cultured for induction for 6 h; 7: The precipitation of the sample containing pET-28a empty vector cultured for induction for 2 h; 8: The precipitation of the sample cultured for induction for 2 h without IPTG
图 3 Cbl蛋白的纯化图谱
M:低分子质量蛋白Marker(14.4~97.4 kDa);1:超声破碎后的上清;2:镍柱孵育后的过柱蛋白;3:50 mM咪唑洗脱蛋白;4:100 mmol·L−1咪唑洗脱蛋白;5:150 mmol·L−1咪唑洗脱蛋白;6:200 mmol·L−1咪唑洗脱蛋白;7:300 mmol·L−1咪唑洗脱蛋白;8:400 mmol·L−1咪唑洗脱蛋白;9:500 mmol·L−1咪唑洗脱蛋白。
Fig. 3 Purification map of Cbl protein
M: Protein Marker (14.4-97.4 kDa); 1: Supernatant after ultrasonic crushing; 2: Over-column proteins after Ni column incubation; 3: Protein eluted with 50 mmol·L−1 imidazole; 4: Protein eluted with 100 mmol·L−1 imidazole; 5: Protein eluted with 150 mmol·L−1 imidazole; 6: Protein eluted with 200 mmol·L−1 imidazole; 7: Protein eluted with 300 mmol·L−1 imidazole; 8: Protein eluted with 400 mmol·L−1 imidazole; 9: Protein eluted with 500 mmol·L−1 imidazole.
表 1 引物序列
Table 1 Primer sequence
引物 Primers 核苷酸序列(5′→3′) Nucleotide sequence (5′→3′) 长度/bp Length cbl-F CCGGAATTCGTGAATTTTCAGCAGTTACG 29 cbl-R CCCAAGCTTTCAAATCTGGAAGTCGAG 27 -
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