CN110591935A - 地衣芽孢杆菌BL3的Sir2样蛋白制备、纯化及应用 - Google Patents
地衣芽孢杆菌BL3的Sir2样蛋白制备、纯化及应用 Download PDFInfo
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Abstract
本发明公开了一种具有去乙酰化酶活性且依赖于NAD+的地衣芽孢杆菌BL3的Sir2样蛋白的制备、纯化及其应用,属于生物技术领域。本发明具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的地衣芽孢杆菌BL3。本发明经实验发现,分离纯化的地衣芽孢杆菌BL3 Sir2样蛋白具有较强的去乙酰化酶活性,其荧光活性强度约为对照组的30倍,因此认为地衣芽孢杆菌BL3 Sir2样蛋白是一种依赖于NAD+的去乙酰化酶,可调控肝细胞脂质代谢,阻碍脂肪蓄积,抑制肝细胞脂肪变性。
Description
技术领域
本发明属菌种蛋白活性筛选和应用机制的生物技术领域,更具体地说,本发明涉及一种具有去乙酰化酶活性且依赖于NAD+的地衣芽孢杆菌BL3的Sir2样蛋白的制备、纯化及其应用。
背景技术
Sir2蛋白家族(Sirtuin)是一种保守蛋白,它主要作为NAD+依赖性蛋白去乙酰化酶,其存在于包括古细菌到哺乳动物各种生物中。研究表明,Sirtuins广泛参与调节机体能量代谢调节,保持机体糖脂代谢稳态,其中以SIRT1、SIRT3、SIRT6研究的最为广泛。SIRT1是该家族研究最多的一个成员,当前的研究已经表明其不但可以调控组蛋白的去乙酰化作用,而且还对非组蛋白也有很好的调节作用。在体内和体外SIRT1可通过PGC-1α转录因子来调节糖异生、糖酵解,导致线粒体数量和功能的增加。另外有研究表明,SIRT1可以改善线粒体功能障碍,当过表达SIRT1时改善高糖诱导的骨骼肌细胞胰岛素抵抗,进一步研究发现这是通过调节SIRT1-SIRT3-线粒体的途径来实现的。在肌肉中,SIRT1能够通过去乙酰化PGC1-α来增强线粒体中的脂肪酸氧化。另外SIRT3是最主要的一种线粒体去乙酰化酶,它的很多靶蛋白已被确定,其中有许多参与调节重要的代谢平衡。此外,SIRT3去乙酰化复合体I、II、III,它们参与氧化磷酸化(OXPHOS),线粒体有氧呼吸的最后阶段。在HEK293T细胞内过表达线粒体sirtuin蛋白改变糖酵解和线粒体功能。其次,SIRT6参与基因组DNA的稳定和修复代谢和衰老的作用。已有研究表示SIRT6 缺失小鼠会出现低血糖症状,而在SIRT6缺失的细胞中葡萄糖摄取增加,糖酵解增强而线粒体呼吸功能减弱。总结以上研究,不难发现Sirtuin蛋白与细胞能量代谢密切相关,而且在一定程度上它们充当着糖酵解到氧化代谢的开关。
越来越多的研究表示益生菌可以改善代谢等疾病。益生菌中具有Sir2同源蛋白,但关于Sir2蛋白在益生菌中的生物学功能少有报道。近年来发现肠道菌群的变化与非酒精性脂肪性肝病(NAFLD)的发生有着极为重要的关联性,研究表明,益生菌与胃肠道感染、细菌移位、胆固醇代谢等有着直接联系。地衣芽孢杆菌(Bacillus licheniformis)BL3是一种革兰氏阳性(G+)、兼性厌氧、孢子形成的益生菌,俗称整肠生,是一种存在多种特殊功能的菌株,能产生多种酶及蛋白等物质,在畜牧业、农业、医药、食品等方面均有极为广泛的研究价值。地衣芽孢杆菌以活菌制剂对疾病发挥着防治作用,且对生物体自身无任何伤害。因此可利用其对人体无害,甚至还促进人体中外来细菌或过盛细菌的拮抗作用,通过细菌对细菌的抑制作用和生物拮抗作用达到预防和控制的目的并提高健康水平。因为地衣芽孢杆菌是一种兼性厌氧菌,可以发挥生物夺氧的作用,使益生菌得到更好的生长环境,维持肠道中的生态平衡。
目前的研究已经表明哺乳动物Sirtuin蛋白家族可以调节细胞代谢,包括细胞呼吸、脂肪酸代谢等。Sirtuins正常活性的维持有助于延缓,甚至阻止代谢相关疾病的发生。以往对于哺乳动物Sirtuin蛋白家族的研究,表明其广泛参与糖脂代谢的调控及维持能量代谢平衡,并且有望成为代谢疾病的靶点。但就目前的研究来说,有关益生菌Sir2同源蛋白生物学功能却少有报道,益生菌Sir2样蛋白的活性机制尚不明确。且研究发现的以代谢紊乱等为诱发原因的NAFLD,到目前为止仍无特效药物与治疗策略。因此,有必要作进一步研究。
发明内容
本发明筛选出具有去乙酰化酶活性且依赖于NAD+的地衣芽孢杆菌(Baclicus lincheniformis) BL3Sir2样蛋白并研究其活性强弱,使其在疾病治疗中发挥作用。
本发明具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的地衣芽孢杆菌(Baclicus lincheniformis) BL3,提供了一种来源于地衣芽孢杆菌BL3的具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备和作用机制,主要是Bli-sir2基因的重组和Bli-Sir2样蛋白的制备、分离、纯化、去乙酰化酶活性鉴定方法。
本发明还提供了一种去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备方法,其特征在于,重组质粒pET-28a(+)-Bli-sir2的构建及其应用,其构建图谱如图1所示。
本发明还提供了一种去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备方法,包括包涵体的制备及其应用。
本发明还提供了一种包含去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的食品,其特征在于,包括有效剂量的作为活性成分的权利要求1所述具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的地衣芽孢杆菌Baclicus lincheniformis和/或其菌体蛋白成分,且可用于静脉注射。
本发明还提供了一种包含去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的药物,其特征在于,包括有效剂量的作为活性成分的权利要求1所述具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的地衣芽孢杆菌Baclicus lincheniformis和/或其菌体蛋白成分,且可用于静脉注射。
附图说明
图1为实施例1 pET-28a(+)-Bli-sir2真核表达载体的构建图谱。
图2为实施例1目的蛋白表达鉴定结果,其中M为marker,1为经IPTG诱导,2为未经IPTG诱导。
图3为实施例1 SDS-PAGE分析Baclicus lincheniformis Sir2 (Bli-Sir2)蛋白的纯化结果,其中M为marker,1为全蛋白离心上清液,2为洗涤缓冲液A洗脱液,3为洗涤缓冲液B洗脱液,4为结合缓冲液洗脱液。
图4为实施例1 BCA法对纯化的蛋白进行浓度测定的蛋白标准曲线。
图5为实施例1 Baclicus lincheniformis Sir2 (Bli-Sir2)蛋白去乙酰化活性的荧光强度的测定结果,其中Control A为Bli-Sir2 (-) + NAD+ (+),Control B为Bli-Sir2 (+) + NAD+ (-),Bli-Sir2为Bli-Sir2 (+) + NAD+ (+),图中数值为均值±标准误;与Control B组相比,***p < 0.001。
图6为实施例1 构建的pEGFP-N1-Bli-sir2真核表达载体质粒图谱。
图7为实施例1 pEGFP-N1-Bli-sir2双酶切琼脂糖凝胶电泳鉴定结果图。
图8为实施例1免疫印迹法检测重组质粒Bli-Sir2蛋白表达图,其中1为未转染组;2为pEGFP-N1空质粒转染组;3为pEGFP-N1-Bli-sir2重组质粒转染组。
图9为实施例1不同肝细胞油红O染色图像(放大倍数为200倍),其中Control为正常大鼠肝细胞对照组;Model为NAFLD大鼠肝细胞的模型组;Vector为NAFLD大鼠肝细胞转染pEGFP-N1的空质粒组;Bli-Sir2为NAFLD大鼠肝细胞转染pEGFP-N1- Bli-sir2的重组质粒组。
图10为实施例1Bli-Sir2蛋白影响大鼠肝细胞丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性水平的柱状图。
图11为实施例1 Bli-Sir2蛋白影响大鼠肝细胞甘油三酯(TG)和总胆固醇(TC)含量的柱状图。
具体实施方式
以下结合实施例,对本发明进行进一步详细说明。
实施例1:地衣芽孢杆菌Bli-Sir2蛋白的制备及纯化
1. 实验材料
1. 1菌株、质粒
表1 菌株信息
菌株(命名) | 特性描述 | 来源 |
<i>B. licheniformis </i>ATCC 14580 | 野生型 | 本实验室保藏 |
<i>E. coli</i> DH5α | 克隆受体菌 | 本实验室保藏 |
<i>E. coli </i>BL21 (DE3) | 表达宿主菌 | 本实验室保藏 |
表2 质粒信息
质粒(命名) | 遗传标记及说明 | 来源 |
pET-28a(+)-Bli-sir2 | Kan<sup>r</sup>;6×His-tag;携带Bli-sir2(<i>npdA</i>)基因 | 大连宝生物工程有限公司构建 |
1.2主要培养基及试剂
(1)LB培养基:酵母提取物0.5 g,胰蛋白1.0 g,氯化钠1.0 g,加入蒸馏水定容100 mL,水浴溶解,调至pH 7.0,121℃高压蒸汽灭菌20 min;固体培养基则加入2 g琼脂后灭菌。
(2)IPTG:100 mM IPTG(异丙基硫代-β-D-半乳糖苷):2.38 g IPTG溶于100 mLddH2O中,0.22 μm滤膜抽滤,- 20℃保存。
(3)Washing Buffer A:3.5 mmol/L imidazole,0.5 mol/L NaCl,20 mmol/LTris-HCl,pH = 8.0。
(4)Washing Buffer B:100 mmol/L imidazole,0.5 mol/L NaCl,20 mmol/LTris-HCl,pH = 8.0。
(5)Elution Buffer:200 mmol/L imidazole,0.5 mol/L NaCl,20 mmol/L Tris-HCl,pH = 8.0。
实验方法
1.3.1重组质粒的构建与鉴定
质粒pET-28a(+)-Bli-sir2由大连宝生物工程有限公司构建。质粒构建的原理如图1所示。
E. coliα感受态细胞制备
(1)将实验室保藏的E. coli DH5α菌种接种于LB平板,37℃培养过夜;
(2)挑取单菌落接种于50 mL LB液体培养基中,37℃培养,至OD值为0.4 ~ 0.6左右;
(3)移取25 mL菌液至预冷过的50 mL EP管中,并在冰上放置30 min,使培养物冷却至4℃,低温4000 rpm离心10 min,弃上清,回收菌体;
(4)加入5 mL预冷的0.1 mol/L CaCl2,重悬沉淀后,冰浴30 min;于4℃ 4000 rpm离心10 min,弃上清,回收菌体;
(5)加入1 mL预冷的0.1 mol/L CaCl2(含15%甘油)重悬菌体沉淀;
(6)在冰上将细胞分装成小份,100 μL/份,- 80℃冻存备用。
重组质粒转化E. coli DH5α
(1)取100 μL E. coli DH5α感受态细胞悬液于1.5 mL离心管中,再加入约10 μL重组质粒,混匀后冰浴30 min;
(2)42℃水浴,热休克90 s,之后迅速冰上冷却2 min;
(3)立即向上述管中加入1 mL的LB液体培养基,摇匀后于37℃、200 rpm振荡培养约45~ 60 min,使受体菌活化;
(4)取50 μL菌液涂布于Amp-LB固体培养基上,晾干后,37℃培养过夜;
(5)挑取白色菌落,接种于5 mL Amp-LB液体培养基上,37℃培养过夜。
重组质粒的提取
根据HP Plasmid Mini Kit I质粒提取试剂盒的说明,进行质粒提取操作。
重组质粒的鉴定
(1)把收集的pEGFP-N1-Bli-sir2重组质粒用EcoR I和BamH I双酶切,37℃水浴,反应液配制如下:
10 × 2 Buffer | 3.0 μL |
pEGFP-N1-Bli-sir2 | 3.0 μL |
EcoR I | 0.1 μL |
BamH I | 0.1 μL |
DEPC水 | 43.8 μL |
总体积 | 50.0 μL |
(2)制备1.5%琼脂糖胶液:取1.5 g琼脂溶于100 mL TAE电泳缓冲液中,接着微波炉加热至琼脂糖完全融化,待溶液冷却至70℃时,加入1.5 μL 10 mg/mL的EB;
(3)将温热琼脂糖倒入胶槽中,冷却直至成凝胶(凝胶的厚度在3 ~ 5 mm之间),凝固约30 min;
(4)在凝胶完全凝固之后,小心移去梳子,放入电泳槽内,加样孔一侧靠近阴极,注入适量的1 × TAE电泳缓冲液,通常缓冲液高于胶面1 cm;
(5)将适量质粒样品与上样缓冲液(溴酚蓝)混合,用移液枪将样品加入加样孔,之后连接电泳槽和电源,设定电压100 V,当溴酚蓝移动至距离胶板下沿1 cm处时,停止电泳;
(6)取出凝胶,用含有0.5 μg/mL的EB/1 × TAE溶液染色30 min,再用蒸馏水清洗15min;
(7)在紫外灯下,310 nm波长处观察染色后的凝胶,并用凝胶成像系统拍照保存。
重组质粒的纯度和含量测定
用紫外分光光度法测定所提质粒的纯度和浓度:将一定体积的质粒DNA溶液用移液器吸取,在紫外分光光度计上分别检测230 nm、260 nm和280 nm波长下的吸光度A230、A260和A280,计算A260/A280和A260/A230比值,并计算样品的浓度与纯度,计算公式如下:
DNA样品的浓度(μg/μL)= (A260 × 稀释位数 × 50)/1000。
Baclicus lincheniformis Sir2 蛋白的诱导表达
将转染pET-28a(+)-Bli-sir2重组质粒的E. coli BL21取出并活化,划平板挑取单菌落,接种于5 mL Kan-LB培养基上,37℃下振荡培养至OD600 0.5左右,取10 μL样品用于SDS-PAGE分析。接着在剩余菌体中加入IPTG使其浓度为1.0 mM,诱导培养4 h,收集菌体,取10 μL样品用于Tricine SDS-PAGE分析。
E. coli菌体发酵
(1)取出保存的已鉴定的阳性菌株500 μL,无菌条件下接入50 mL Kan-LB培养基上,于37℃下200 rpm振荡培养过夜;
(2)将活化的种子无菌接入发酵罐,再加入30 mL卡那霉素溶液,控制空气流量在0.5vvm,罐压在0.04 ~ 0.05 mpa,溶氧保持在40%以上;
(3)发酵3 h后,每隔1个小时取样检测发酵液的OD600,当OD600 ≥ 0.8时,加入1 mol/L诱导剂IPTG,诱导剂的终浓度为1 mM,过程中控制溶氧不低于40%;
(4)诱导表达9 h后,发酵结束,放罐,发酵液5000 rpm离心20 min,收集菌体,- 20℃冷冻保存。
包涵体的制备
(1)取收集的菌体25 g,加入250 mL Washing Buffer A,混匀后,用超声粉碎仪粉碎细胞;
(2)将粉碎后的细胞分装到50 mL EP管中,在4 ℃下8500 rpm离心30 min,弃去上清液,收集沉淀;
(3)将沉淀用Washing Buffer A稀释,再分装至50 mL EP管中,超声粉碎仪超声悬浮,静置10 min后,4℃下8500 rpm离心30 min,弃上清,收集沉淀;
(4)重复第3步一次;
(5)用50 mL Washing Buffer A超声悬浮包涵体,洗去残留的triton和EDTA,4℃下8500 rpm离心30 min,收集的沉淀即为包涵体。
Baclicus lincheniformis Sir2蛋白的分离和纯化
(1)NTA层析柱准备:向层析柱中加入1 mL NTA介质,并分别依次用纯化水和WashingBuffer A各洗涤一次;
(2)取1 mg包涵体,在冰浴中冻融菌体沉淀,加入10 mL Washing Buffer A悬浮,超声助溶,室温下放置1 h后,4℃下12000 rpm离心30 min,收集上清液;
(3)上清样品以0.5 mL/min流速上Ni2+-NTA柱,收集流出液,取10 μL样品用于SDS-PAGE分析;
(4)用Washing Buffer A和Washing Buffer B依次洗脱杂蛋白,速度控制在0.5 mL/min,收集流出液,取10 μL样品用于SDS-PAGE分析;
(5)用Elution Buffer洗脱目标蛋白,速度控制在0.5 mL/min,收集洗脱峰,取10 μL样品用于SDS-PAGE分析。
分析蛋白纯度及分子量
(1)配制12%的分离胶:30%的丙烯酰胺4.0 mL,10% SDS 0.1 mL,1.5 mol/L Tris-HCl2.5 mL,10%过硫酸铵50 μL,TEMED 8 μL,蒸馏水3.3 mL;
(2)配制5%的浓缩胶:30%的丙烯酰胺830 μL,10% SDS 50 μL,1.0 mol/L Tris-HCl630 μL,10%过硫酸铵50 μL,TEMED 10 μL,蒸馏水3.4 mL;
(3)在胶板中加入6 mL分离胶,并用适量无水乙醇液封,待分离胶凝固后,倒掉上层无水乙醇;
(4)倒入浓缩胶,插上梳子,静置30 min,凝胶完全凝固;
(5)拔出梳子,在电泳槽中加入注入适量3 mol/L Tris-HCl电泳缓冲液,缓冲液高于胶面1 cm;
(6)将10 μL的蛋白样品与上样缓冲液(0.12 mol/L Tris-HCl:5%甘油、2% SDS、0.1%溴酚蓝、3 mmol/L 巯基乙醇)混匀,100℃水浴5 min,再用移液枪将样品加入加样孔;
(7)连接电泳槽和电源,先设定电压50 V,电泳30 min,再设定电压120 V,电泳90 min;
(8)取出凝胶,用含有考马斯亮蓝R-250染色20 min,再脱色;
(9)用凝胶成像系统拍照保存,分析蛋白纯度及分子量。
法测定蛋白浓度
根据BCA Protein Assay Kit蛋白质定量试剂盒操作说明,进行蛋白质定量,根据标准曲线即可计算出蛋白质的浓度。
纯化蛋白的去乙酰化酶活性鉴定
(1)实验分三组,首先配制实验组反应液:Tris-HCl缓冲液25 mM,KCl 2.7 mM,NaCl100 mM,MgCl2 1 mM,NAD+ 500 μM,Z-(Ac)Lys-AMC 8 μL,纯化后的Bli-Sir2蛋白20 μg;对照组A反应液的配制基本同实验组,但不加Bli-Sir2蛋白,用等量纯化水代替;对照组B反应液的配制基本同实验组,但不加NAD+,用等量纯化水代替。
(2)将两组反应液皆避光静置,控制在37℃下水浴10 h;
(3)向两组反应物中分别加入70 μg/mL胰蛋白酶70 μL,继续反应1 h;
(4)反应结束后,分别将两组反应液移至两块黑色的96孔板中,用酶标仪分别测定其荧光值(λem=340 nm;λex=460 nm)。
统计学分析
使用SPSS18.0软件处理数据,两组比较作t检验,多组均数间比较采用One-way ANOVA分析,显著性差异用p<0.05或p<0.01或p<0.001表示。
实验结果
2.1 Bli-Sir2蛋白的诱导表达
收集诱导表达的菌体,用Tricine SDS-PAGE检测目的蛋白的表达,结果如图2所示。在第1泳道有条约29 kDa的粗条带,而未用IPTG诱导的第2泳道中无相应条带。
蛋白的纯化
使用E. coli BL21菌株大量表达Bli-Sir2蛋白,得到的蛋白是以包涵体的形式存在。为了得到纯度较高的目的蛋白,我们将制备的包涵体用Ni柱进行亲和层析。由于Ni柱中的氯化镍可以和含有His标签的融合蛋白结合,因此可与我们的目的蛋白结合使其纯化。对洗脱液进行SDS-PAGE,结果如图3所示,最后洗脱出来的在29 kDa处有一清晰的条带,其大小与目的蛋白加上His标签的总量基本一致,因此可推测E. coli BL21表达的蛋白是His-Bli-Sir2融合蛋白。通过灰度分析,得到的蛋白纯度为94.7%。
蛋白浓度测定
采用BCA法对纯化的蛋白进行浓度测定,根据标准曲线(图4),最后计算出本实验得到的蛋白浓度为3.1 mg/mL。
蛋白的去乙酰化活性测定
乙酰化荧光底物Z-(Ac)Lys-AMC上的AMC偶联时不会被激发荧光,但当其游离时会被激发荧光,因此,当Z-(Ac)Lys-AMC上的赖氨酸被去乙酰化后,胰蛋白酶可以将连接于赖氨酸上的AMC剪切下来,通过检测游离AMC的荧光强度,即可得到去乙酰化酶的活性。结果如图5所示,相比于未加Bli-Sir2蛋白的对照组A和未加NAD+的对照组B, Bli-Sir2组的荧光强度显著提升,并且约是对照组B的30倍,这表明纯化得到的Bli-Sir2蛋白具有去乙酰化酶活性,且依赖于NAD+。
实施例2 地衣芽孢杆菌BL3 Sir2样蛋白对肝细胞脂肪代谢的影响
1. 实验材料与方法
1.1实验菌株、质粒和细胞
表3 菌株信息
菌种(命名) | 特性描述 | 来源 |
<i>B.licheniformis</i> SRCM100027 | 野生型 | 本实验室保藏 |
<i>E. coli</i> DH5α | 受体菌 | 本实验室保藏 |
表4 质粒信息
表5 细胞株信息
1.2 NAFLD细胞模型的构建
将大鼠正常肝细胞BRL-3A放入含有10%胎牛血清的MEM培养基中,置于25 mL培养瓶内,于5% CO2的培养箱中37℃下培养24 h。当细胞达到70% ~ 80%融合时,加入40 μg/mL油酸使其变性,诱导24 h。第二天换液后再加入油酸,诱导72 h后,收集细胞。
质粒的构建
质粒pEGFP-N1-Bli-sir2由大连宝生物工程有限公司构建,其中地衣芽孢杆菌sir2样基因npdA的信息如下表6所示,质粒构建的原理如图6所示。
表6 地衣芽孢杆菌sir2样基因npdA信息表
Name | Locus | Length (bp) | Protein name |
地衣芽孢杆菌<i>sir2</i>样基因<i>npdA </i>(Bli-sir2) | ARW53201 | 750 | NAD-dependent protein deacetylase |
分别构建E. coli DH5α和E. coli BL21感受态细胞,并先用重组质粒pEGFP-N1-Bli-sir2转化E. coli DH5α感受态细胞,提取质粒并鉴定后,再用重组质粒pEGFP-N1-Bli-sir2转化E. coli BL21感受态细胞,其具体步骤参照实施例1中“1.3.1.1”、“1.3.1.2”、“1.3.1.3”、“1.3.1.4”、“1.3.1.5”。
脂质体转染
1.4.1 重组质粒pEGFP-N1-Bli-sir2转染NAFLD大鼠肝细胞
根据Lipofectamine 2000脂质体转染试剂盒步骤,进行转染操作。
空质粒pEGFP-N1转染NAFLD大鼠肝细胞
步骤同上步实施例2“1.4.1”。
鉴定目的蛋白的表达
1.5.1细胞全蛋白提取
(1)肝细胞BRL-3A传代至60 mm培养皿,待细胞完全融合时,收集细胞;
(2)弃去培养基,加入约2 mL PBS清洗培养皿,弃去PBS,加入2 mL 0.05%胰酶,37℃下孵育1 min;
(3)待细胞变形后,将细胞吹下并转移至1.5 mL离心管中,4℃下10000 rpm离心3 min,弃上清液;
(4)约1 mL PBS清洗沉淀,4℃下10000 rpm离心3 min;
(5)PBS清洗,弃上清,- 80℃冻存待用;
(6)将细胞沉淀敲散,加入200 μL RIPA缓冲液,吹打沉淀使其松散,冰上放置40 min;
(7)在4℃下10000 rpm离心15 min,上清液即为蛋白,- 80℃冻存。
蛋白定量
根据BCA Protein Assay Kit蛋白质定量试剂盒操作说明,进行蛋白质定量,根据标准曲线即可计算出蛋白质的浓度。
检测蛋白含量
(1)配制10%的分离胶,灌胶时均匀注入,上层加适量无水酒精,室温静置30 min后,倒掉上层液体,灌注4%浓缩胶,插上梳子,静置30 min;
(2)取出40 μg蛋白样品,用PBS溶解,加入20 μL 3×loading buffer和6 μL 1 M的DTT,混匀后,置于100℃中水浴10 min使蛋白变性,将电泳液倒入槽中,每孔道各加10 μL样品,并在两边孔道各加5 μL蛋白marker,50 V恒压电泳;
(3)电泳结束后,用胶铲取出电泳后的胶,浸泡于转膜液中,剪取合适大小的滤纸和PVDF膜,用甲醇泡PVDF膜5 min,再放入转膜液中泡10 min,去除膜上的蜡,然后将“海绵→6层滤纸→凝胶→PVDF膜→6层滤纸→海绵”按从负极到正极的顺序依次组装转印夹层,夹板组装后转移至转膜槽,150 mA恒流90 min;
(4)转膜结束后,用5%脱脂奶粉(0.5 g的脱脂奶粉,用10 mL的TBST溶液溶解)封闭1 h;
(5)孵育一抗:加入用TBST一定比例下稀释的一抗,4℃下孵育过夜;
(6)孵育二抗:第二天将膜取出,复温1 h后用TBST液洗膜3次,每次10 min;加入TBST一定比例稀释的并用HRP标记的二抗,孵育1 h;
(7)将孵育后的PVDF膜取出,用TBST液洗膜3次,每次5 min,然后加入发光液孵育3min,再用滤纸吸干,将胶片和PVDF膜放入压片盒中压片,1 min后取出胶片,定影30 s,用水清洗,烘干,扫描。
细胞油红O染色
(1)将肝细胞悬液接种于12孔板中,置于5% CO2培养箱中,在37℃下培养;
(2)待细胞达到70% ~ 80%的细胞密度时,吸去培养基,用PBS清洗3次,每次5 min;
(3)加入4%多聚甲醛,固定20 min;
(4)吸去固定液并加入油红O工作液,染色20 min后吸去油红O染液;
(5)PBS洗3次,加入60%的异丙醇,清洗5 min以洗去背景色,置于载玻片上,于显微镜下观察并拍照。
肝细胞内培养上清液中ALT、AST活力的测定
1.7.1 ALT活力的测定
(1)细胞处理
将细胞悬液加入6孔板中,每孔2 mL,置于5% CO2培养箱中在37℃下培养36 h后,弃上清液,改用无血清培养基继续培养6 h。超声粉碎细胞,4000 rpm离心15 min吸取上清,收集上清液细胞。
(2)标准曲线测定
将测定所用试剂(除NaOH溶液外)全部置于水浴箱内,预热至37℃。
取6支试管,按下表配制丙氨酸氨基转移酶(ALT)标准溶液,冷却后以0为对照用分光光度计比色测定吸光值OD520 nm。以丙酮酸含量为纵坐标,吸光值OD520 nm为横坐标绘制标准曲线。
表5 ALT标准溶液的配制
试管编号 | 0 | 1 | 2 | 3 | 4 | 5 |
20 μmol/L丙酮酸钠标准溶液/mL | 0.00 | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 |
ALT基质液/mL | 0.50 | 0.45 | 0.40 | 0.35 | 0.30 | 0.25 |
0.1 mol/L磷酸缓冲液/mL | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 |
2,4-二硝基苯肼/mL | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
0.4 mol/L NaOH溶液/mL | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 |
(3)ALT活力的测定
将测定所用试剂除氢氧化钠溶液外全部置于水浴箱内,预热。取2支试管,按下表配制,冷却后以0为对照用分光光度计比色测定吸光值OD520 nm。在标准曲线中读出样品丙酮酸的生成量。
表6 ALT活力的测定溶液配制表
试管编号 | 1 | 2 |
样品/mL | 0.10 | 0.10 |
ALT基质液/mL | - | 0.50 |
ALT基质液/mL | 0.50 | - |
2,4-二硝基苯肼/mL | 0.50 | 0.50 |
0.4 mol/L NaOH溶液/mL | 5.00 | 5.00 |
1.7.2 AST活力的测定
(1)细胞处理
同实施例2“1.12.1”中的“细胞处理”方法一致。
(2)标准曲线测定
将测定所用试剂除氢氧化钠溶液外全部置于水浴箱内,预热。取6支试管,按下表配制天冬氨酸氨基转移酶(AST)标准溶液,冷却后以0管为对照用分光光度计比色测定吸光值OD520 nm。以丙酮酸含量为纵坐标,吸光值OD520 nm为横坐标绘制标准曲线。
表7 AST标准溶液配制表
试管编号 | 0 | 1 | 2 | 3 | 4 | 5 |
20 μmol/L丙酮酸钠标准溶液/mL | 0.00 | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 |
AST基质液/mL | 0.50 | 0.45 | 0.40 | 0.35 | 0.30 | 0.25 |
0.1 mol/L磷酸缓冲液/mL | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 |
2,4-二硝基苯肼/mL | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
NaOH溶液/mL | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 |
(3)AST活力的测定
同实施例2“1.12.1”中的“ALT活力的测定”方法一致。
检测肝细胞内TG含量
根据大鼠甘油三酯(TG)ELISA试剂盒操作说明,进行肝细胞内TG含量测定操作。
检测肝细胞内TC含量
根据总胆固醇(TC)测定试剂盒操作说明,进行TC含量测定操作。
统计学分析
使用SPSS18.0软件处理数据,两组比较作t检验,多组均数间比较采用One-way ANOVA分析,显著性差异用p<0.05或p<0.01或p<0.001表示。
实验结果
2.1重组质粒pEGFP-N1-Bli-sir2的鉴定
提取的重组质粒pEGFP-N1-Bli-sir2经EcoR I和BamH I双酶切,酶切产物通过琼脂糖凝胶电泳鉴定,结果如图7所示,在泳道1上检测到约4700 bp的载体带和约750 bp的Bli-sir2基因带,与预期的结果一致。将酶切正确的菌液送至大连宝生物工程有限公司测序,将检测结果与目的序列进行比对,结果完全一致。
重组质粒Bli-Sir2蛋白表达的鉴定
Bli-Sir2蛋白没有特异性抗体,因此我们通过检测anti-EGFP的特异性抗体对Bli-Sir2蛋白的表达进行鉴定。结果如图8所示,发现第3泳道有约55 kDa的条带,这符合EGFP-Bli-Sir2融合蛋白的分子量,说明重组质粒pEGFP-N1-Bli-sir2可以在大鼠NAFLD肝细胞中表达。同时观察到泳道2上有大约27 kDa的条带,说明空质粒pEGFP-N1在NAFLD大鼠肝细胞中也能正常表达。
油红O染色鉴定结果
NAFLD发生的重要表现之一是肝细胞脂肪变性和肝纤维化。当肝细胞内出现大小不等的空泡脂滴时,说明发生了脂肪变性。因此抑制肝细胞脂肪变性可在一定程度上阻碍NAFLD的发生。
通过油红O染色对肝细胞进行形态学观察,发现正常大鼠肝细胞的细胞边缘清晰,结合紧密,胞浆丰富,细胞内几乎无红色脂滴存在。而用油酸诱导的NAFLD模型组肝细胞边缘模糊,胞间存在空隙,有较多的红色脂滴存在。这表明通过油酸诱导可以成功且有效地建立NAFLD细胞模型。同时,我们对比发现转染重组质粒pEGFP-N1-Bli-sir2的NAFLD肝细胞相比于空质粒组中的脂质蓄积程度有明显的改善,红色脂滴较模型组明显减少,细胞轮廓变清晰。这些结果表明Bli-Sir2蛋白可以显著抑制NAFLD细胞的脂肪变性(图9)。
对大鼠肝细胞AST和ALT水平的影响
ALT和AST都是常用于肝功能检查的指标,当肝细胞损伤时,细胞内的转氨酶就会被释放,从而使ALT和AST的酶活性升高。我们分别检测了四组细胞的ALT及AST酶活性,结果如图10所示,与Control组相比,NAFLD模型组的AST及ALT水平明显升高,有统计学差异。相对于NAFLD+Vector组,NAFLD+Bli-Sir2组的AST及ALT水平均显著下降,有统计学意义。说明Bli-Sir2有助于缓解NAFLD肝细胞损伤。
对大鼠肝细胞TC和TG水平的影响
当肝脏发生脂肪变性时,脂质的代谢会失调,而蓄积在肝细胞中,因此我们通过测定肝细胞中TG和TC的含量来评价肝细胞脂质代谢水平。由图11中可以看出,NAFLD模型组的TC和TG含量较对照组均明显增加,再次验证了NAFLD的肝细胞模型建立成功。而在转染Bli-Sir2的NAFLD细胞中,TC和TG含量有不同程度的降低,且均具有显著性。而TG和TC含量在转染空载体的NAFLD细胞与模型组的NAFLD细胞中无特别差异。以上结果说明了Bli-Sir2可以明显减少TG和TC的聚集,调控脂质的代谢过程。
Claims (6)
1.一种来源于地衣芽孢杆菌(Baclicus lincheniformis)BL3的具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备和作用机制。
2.权利要求1所述的一种来源于地衣芽孢杆菌BL3的具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备和作用机制,其特征在于Bli-sir2基因的重组和Bli-Sir2样蛋白的制备、分离、纯化、去乙酰化酶活性鉴定。
3. 权利要求2所述的 Bli-sir2基因的重组和Bli-Sir2样蛋白的制备、分离、纯化、去乙酰化酶活性鉴定,其特征在于,SDS-PAGE分析呈现清晰条带,且灰度分析知蛋白纯度为95.7%以上,浓度为3.1 mg/mL以上,测序结果同理论值。
4.权利要求1所述的一种来源于地衣芽孢杆菌BL3的具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备和作用机制,其特征在于,游离AMC荧光强度分析显示所得纯化的Bli-Sir2蛋白具有去乙酰化酶活性,且约为对照组的30倍。
5.权利要求1所述的一种来源于地衣芽孢杆菌BL3的具有去乙酰化酶活性且依赖于NAD+的Sir2样蛋白的制备和作用机制,其特征在于,所制备的Bli-Sir2样蛋白具有调控肝细胞脂质代谢,阻碍脂肪蓄积和抑制肝细胞脂肪变性的作用。
6.权利要求5所述的所制备的Bli-Sir2样蛋白具有抑制肝细胞脂肪变性的作用,其特征在于,Bli-Sir2样蛋白具有调控肝细胞脂质代谢,阻碍脂肪蓄积和抑制肝细胞脂肪变性所致的脂肪肝、肝纤维化、肝硬化等的食品或药品中的应用。
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