CN107022534B - 长牡蛎胞壁水解酶重组蛋白的制备及制备获得的重组蛋白的应用 - Google Patents
长牡蛎胞壁水解酶重组蛋白的制备及制备获得的重组蛋白的应用 Download PDFInfo
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Abstract
本发明属于分子生物学和生物化学技术领域，具体涉及一种来自长牡蛎胞壁水解酶重组蛋白的制备及制备获得的重组蛋白的应用。利用酵母表达体系获得长牡蛎胞壁水解酶的重组蛋白。本发明利用酵母表达体系获得了长牡蛎胞壁水解酶的重组蛋白对大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌、灿烂弧菌、创伤弧菌、溶藻弧菌、恶臭假单胞菌和铜绿假单胞菌的生长具有显著的抑制作用。本发明获得的重组蛋白可用于抑菌剂开发。
Description
技术领域
本发明属于分子生物学和生物化学技术领域，具体涉及一种来自长牡蛎胞壁水解酶重组蛋白的制备及制备获得的重组蛋白的应用。
背景技术
长牡蛎是世界重要的海水养殖贝类之一，在我国水产养殖经济中长期占据举足轻重的地位。近年来，由各种病原微生物引起的疾病在长牡蛎的养殖群体中不断爆发，对养殖产业造成了巨大损失。因此，对长牡蛎开展免疫防御机制研究，发掘有效的抗病相关功能蛋白将有助于人类对其病害进行有效防控。
长牡蛎隶属于软体动物，缺乏适应性免疫系统，仅依赖固有免疫系统抵御病原入侵。抗菌肽/蛋白作为固有免疫中重要效应分子，直接参与了对病原微生物的杀伤和清除过程。通过生物信息学技术分析发现，长牡蛎基因组序列编码了若干个胞壁水解酶基因。其中，CgCLE-1(Genbank注册号：XP_011436111.1)基因开放阅读框为501bp，其编码的蛋白含有一段120个氨基酸的Hydrolase_2结构域。CgCLE-1氨基酸序列除与长牡蛎中其他胞壁水解酶序列一致性为55-99％，与梭状芽孢杆菌属Clostridium、芽孢杆菌属Bacillus细菌的细胞壁水解酶(cell wall hydrolase)或孢子皮质裂解酶(spore cortex-lytic enzyme)一致性约为30-40％。目前，国内外对细菌来源的胞壁水解酶进行了功能研究，发现其可特异性地降解孢子皮质中的肽聚糖，在孢子萌发过程发挥重要作用。但对无脊椎动物胞壁水解酶的制备、功能及其应用的研究尚未见报道。本发明利用酵母表达体系制备长牡蛎胞壁水解酶CgCLE-1重组蛋白，并对制备获得的重组蛋白进行抑菌功能研究。
发明内容
本发明的目的在于提供一种来自长牡蛎胞壁水解酶重组蛋白的制备及制备获得的重组蛋白的应用。
为实现上述目的，本发明采用的技术方案是：
一种长牡蛎胞壁水解酶重组蛋白的制备，利用酵母表达体系获得长牡蛎胞壁水解酶的重组蛋白。
具体为：
1)通过长牡蛎血淋巴细胞总RNA，反转获得cDNA；
2)在CgCLE-1的Hydolase_2结构域编码区两端分别设计含有限制性酶切位点的引物；
序列为：5’-GAATTCAGAGGAGAACCGATGGAAGCTA-3’，5’-GCGGCCGCCACAAAGTACAGACCCCCGAGTT-3’；
3)以步骤1)获得cDNA为模板，采用步骤2)的引物通过PCR扩增获得该基因片段，并将其克隆到pPICZαA表达载体中构建重组质粒；
4)所述获得重组质粒经ScaI线性化后，电转入宿主细胞毕赤酵母Pichiapastoris GS115中实现真核体外重组表达，而后纯化获得CgCLE-1重组蛋白。
一种利用制备方法获得长牡蛎胞壁水解酶重组蛋白的应用，所述长牡蛎Crassostrea gigas胞壁水解酶(CgCLE-1)的重组蛋白作为抑菌剂的应用。
所述长牡蛎Crassostrea gigas胞壁水解酶(CgCLE-1)的重组蛋白作为大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌、灿烂弧菌、创伤弧菌、溶藻弧菌、恶臭假单胞菌或铜绿假单胞菌的抑菌剂的应用。
本发明所具有的优点:
本发明对长牡蛎胞壁水解酶CgCLE-1(Accession NO.XP_011436111.1)进行体外真核重组表达，所得长牡蛎胞壁水解酶重组蛋白是通过酵母表达系统所得，既具有生长快、易培养、遗传操作简单等原核表达系统的特点，又具有真核生物对蛋白的加工、修饰和空间折叠等功能，利于活性蛋白的表达。其对水生生物常见致病菌大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌、灿烂弧菌、创伤弧菌、溶藻弧菌、恶臭假单胞菌和铜绿假单胞菌的生长均有明显的抑制效果，在水生生物抗菌剂方面具有潜在应用价值。
附图说明
图1为本发明实施例提供的长牡蛎CgCLE-1重组蛋白电泳图。
图2为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对灿烂弧菌生长的抑制作用效果图。
图3为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对大肠杆菌生长的抑制作用效果图。
图4为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对创伤弧菌生长的抑制作用效果图。
图5为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对恶臭假单胞菌生长的抑制作用效果图。
图6为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对溶藻弧菌生长的抑制作用效果图。
图7为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对铜绿假单胞菌生长的抑制作用效果图。
图8为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对嗜水气单胞菌生长的抑制作用效果图。
图9为本发明实施例提供的长牡蛎CgCLE-1重组蛋白对金黄色葡萄球菌生长的抑制作用效果图。
具体实施方式
下面的实验例中将对本发明作进一步的阐述，但本发明不限于此。
本发明是通过PCR技术克隆获得了长牡蛎胞壁水解酶中Hydolase_2结构域的编码区序列，克隆到pPICZαA表达载体中，在毕赤酵母GS115中实现真核体外重组表达，经镍琼脂糖凝胶纯化后获得的重组蛋白分子，检测重组蛋白的抑菌活性。
实验例1：长牡蛎CgCLE-1体外原核重组表达和纯化
1、重组载体的构建
本发明中采用的重组载体为Invitrogen公司的pPICZαA原核表达载体。从新鲜长牡蛎血淋巴细胞中提取总RNA，并通过反转录PCR获得cDNA。通过PCR技术，采用5’末端分别添加了EcoRI和Not I限制性酶切位点的引物P1(5’-GAATTCAGAGGAGAACCGATGGAAGCTA-3’)和P2(5’-GCGGCCGCCACAAAGTACAGACCCCCGAGTT-3’)扩增长牡蛎胞壁水解酶Hydolase_2结构域的编码区。反应条件为：首先94℃预变性5分钟，然后进入下列循环：94℃变性30秒，65℃退火30秒，72℃延伸2分钟，共进行35个循环，最后72℃延伸10分钟。用胶回收纯化试剂盒(大连宝生物工程有限公司)将扩增片段纯化回收，与pMD19-T载体连接。转化后筛选阳性克隆，提取质粒，并使用EcoRI和Not I对质粒进行双酶切；回收目的片段，并与经EcoRI和NotI双酶切的表达载体pPICZαA连接。经测序验证，插入序列与长牡蛎CgCLE-1中Hydolase_2结构域的编码区序列一致。
2、重组蛋白的表达与纯化
将重组质粒用Sac I酶切，并纯化酶切产物。采用电击法将10μg线性化重组质粒转入毕赤酵母Pichia pastoris GS115菌株，涂布于含10-200μg ml-1Zeocin的YPDS平板，于30℃培养3-4天，筛选高拷贝转化菌株。挑取YPD Zeocin平板上的阳性单菌落，接种于含有10mL BMGY培养基的摇瓶中，28-30℃振荡培养(250-300rpm)直至对数生长期(OD600为2.0-6.0)。1500-3000×g室温离心5min，收集酵母细胞。将酵母细胞重悬于100-200mL BMMY培养基中(OD600为1.0)，28-30℃振荡培养，每隔24小时补加甲醇至终浓度为2％进行诱导。诱导4天后，3000×g 4℃离心5min，收集发酵上清液进行重组蛋白的纯化和浓缩。发酵上清加入超滤离心管(MWCO 30kDa,Millipore)，12000×g 4℃离心30min；流穿液加入超滤离心管(MWCO 3kDa,Millipore)，12000×g 4℃离心30min，获得重组表达蛋白，采用12％Tricine-SDS-PAGE检测。
实验结果：电泳结果表明，得到单一蛋白条带与预测的CgCLE-1分子量大小一致，为纯化的长牡蛎CgCLE-1重组蛋白(图1)。
实验例2：长牡蛎CgCLE-1重组蛋白的抑菌活性检测
大肠杆菌(Escherichia coli)、嗜水气单胞菌(Aeromonas hydrophila和金黄色葡萄球菌(Staphyloccocus aureus)分别用LB培养基37℃，220rpm，培养至对数生长期；灿烂弧菌(Vibrio splendidus)、创伤弧菌(Vibrio vulnificus)、溶藻弧菌(Vibrioalginolyticus)、恶臭假单胞菌(Pseudomonas putida)和铜绿假单胞菌(Pseudomonasaeruginosa)分别用2216E培养基28℃，220rpm，培养至对数生长期。所得菌体分别经离心收集后用TBS(50mM Tris-HCl,100mM NaCl，pH＝7.4)进行洗涤并重悬至浓度104CFU。将50μL上述实施例获得重组蛋白CgCLE-1(160μg mL-1)与等体积的上述获得各细菌重悬液室温孵育2h。空白组(Blank组)以50μL TBS代替重组蛋白。取20μL上述混合物于平底96孔板(Costar，Fisher)中，每孔加入200μL液体培养基，于酶标仪中振荡培养12-16h，每隔30min检测记录各孔OD600值。其中，大肠杆菌、嗜水气单胞菌和金黄色葡萄球菌处理组用LB培养基于37℃振荡培养；灿烂弧菌、创伤弧菌、溶藻弧菌、恶臭假单胞菌、铜绿假单胞菌处理组用2216E培养基28℃震荡培养。每个样品设三个重复，根据3次测定结果取平均值绘制生长曲线并进行统计学分析。
实验结果：CgCLE-1能够显著抑制灿烂弧菌(图2)、大肠杆菌(图3)、创伤弧菌(图4)、恶臭假单胞菌(图5)、溶藻弧菌(图6)、铜绿假单胞菌(图7)、嗜水气单胞菌(图8)和金黄色葡萄球菌(图9)的生长(P<0.01)。实验例3：长牡蛎CgCLE-1重组蛋白最小抑菌浓度(minimum inhibitory concentration,MIC)的测定
将CgCLE-1重组蛋白进行倍比稀释，按照实验例2的方法进行抑菌活性检测，可抑制某种微生物出现明显增长的最低药物浓度即CgCLE-1的MIC。
实验结果：CgCLE-1对所检测细菌的最小抑菌浓度见表1。
表1.CgCLE-1对不同细菌的最小抑菌浓度
Claims (2)
1.一种长牡蛎胞壁水解酶重组蛋白的应用，其特征在于：长牡蛎（Crassostrea gigas）胞壁水解酶CgCLE-1的重组蛋白在制备抑菌剂中的应用；
所述长牡蛎胞壁水解酶CgCLE-1登录号为Accession NO. XP_011436111.1；
长牡蛎胞壁水解酶重组蛋白的制备，利用酵母表达体系获得长牡蛎胞壁水解酶的重组蛋白；
1）通过长牡蛎血淋巴细胞总RNA，反转录 获得cDNA；
2）在CgCLE-1的Hydolase_2结构域编码区两端分别设计含有限制性酶切位点的引物；
序列为：5’- GAATTCAGAGGAGAACCGATGGAAGCTA-3’，5’-GCGGCCGCCACAAAGTACAGACCCCCGAGTT -3’；
3）以步骤1）获得cDNA为模板，采用步骤2）的引物通过PCR扩增获得该基因片段，并将其克隆到pPICZα A表达载体中构建重组质粒；
4）所述获得重组质粒经ScaI线性化后，电转入宿主细胞毕赤酵母（Pichia pastoris）GS115中实现真核体外重组表达，而后纯化获得CgCLE-1重组蛋白。
2.按权利要求1所述的应用，其特征在于：所述长牡蛎（Crassostrea gigas）胞壁水解酶CgCLE-1的重组蛋白在制备抑制大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌、灿烂弧菌、创伤弧菌、溶藻弧菌、恶臭假单胞菌或铜绿假单胞菌的抑菌剂中的应用。
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| CN105647949A (zh) * | 2014-12-08 | 2016-06-08 | 浙江理工大学 | 福寿螺内源性纤维素酶eg27i在毕赤酵母中的组成型表达与纯化 |
| CN105688187A (zh) * | 2015-12-31 | 2016-06-22 | 中国科学院海洋研究所 | 一种长牡蛎C型凝集素-4（CgCLec-4）重组蛋白的应用 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647949A (zh) * | 2014-12-08 | 2016-06-08 | 浙江理工大学 | 福寿螺内源性纤维素酶eg27i在毕赤酵母中的组成型表达与纯化 |
| CN105688187A (zh) * | 2015-12-31 | 2016-06-22 | 中国科学院海洋研究所 | 一种长牡蛎C型凝集素-4（CgCLec-4）重组蛋白的应用 |
| KR20160041037A (ko) * | 2016-04-04 | 2016-04-15 | 김선근 | 굴 복합추출물을 이용한 레토르트파우치 굴국 및 그의 제조방법 |
Non-Patent Citations (5)
| Title |
|---|
| "Identification of three singular glycosyl hydrolase family 18 members from the oyster Crassostrea gigas: Structural characterization, phylogenetic analysis and gene expression";Fabien Badariotti et al.,;《Comparative Biochemistry and Physiology, Part B》;20100922;第158卷;第56-63页 * |
| "PREDICTED: Crassostrea gigas spore cortex-lytic enzyme (LOC105334377)，mRNA，Accession No：XM_011437809.2";Eukaryota et al.,;《GenBank》;20170202;第1页 * |
| "凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测";卞曙光等;《水生生物学报》;20101130;第34卷(第6期);第1091页摘要、第1092页左栏第3段、右栏第1-2段 * |
| Eukaryota et al.,."PREDICTED: Crassostrea gigas spore cortex-lytic enzyme (LOC105334377)，mRNA，Accession No：XM_011437809.2".《GenBank》.2017,第1页. * |
| 卞曙光等."凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测".《水生生物学报》.2010,第34卷(第6期),第1091-1096页. * |
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