CN107988193B - 一种脲基甲酸水解酶及其制备方法 - Google Patents
一种脲基甲酸水解酶及其制备方法 Download PDFInfo
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- CN107988193B CN107988193B CN201711374808.6A CN201711374808A CN107988193B CN 107988193 B CN107988193 B CN 107988193B CN 201711374808 A CN201711374808 A CN 201711374808A CN 107988193 B CN107988193 B CN 107988193B
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- allophanate hydrolase
- ala
- allophanate
- hydrolase
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Abstract
本发明涉及分子生物学领域，尤其涉及一种脲基甲酸水解酶及其制备方法。包括以下步骤：1)利用PCR技术扩增得到脲基甲酸水解酶的克隆，2)构建脲基甲酸水解酶的表达载体；3)将表达载体转化入大肠杆菌感受态菌体中得到重组菌体，进一步诱导表达、和纯化蛋白得到脲基甲酸水解酶。该方法简单，易操作，且发现了新的基因序列的脲基甲酸水解酶，该酶在低温条件下依然具备较强的酶学活性。另外，本发明还提供该脲基甲酸水解酶的多克隆抗体以及其检测方法，对低温菌Cryobacterium属菌种的鉴定提供新的依据，对于脲基甲酸水解酶的研究提供了新的研究方向。
Description
技术领域
本发明属于分子生物学领域，主要涉及一种脲基甲酸水解酶及其应用。
背景技术
脲基甲酸水解酶，英文名：Allophanate hydrolase。脲基甲酸水解酶在三嗪类除草剂的降解过程中起重要作用，它催化降解脲基甲酸，生成氨气和二氧化碳。对于如阿拉特津除草剂等含有三嗪的污染物具有降解作用，在环境保护领域具有广阔的应用前景。
利用脲基甲酸水解酶降解除草剂污染物的中间体脲基甲酸具有极大的应用前景，在低温条件条件下，以脲基甲酸为底物降解生成氨气，净化环境具有较好的效果。
目前生产得到的脲基甲酸水解酶对低温的耐受能力差，在低温下几乎不能发挥活性，在工业生产上，对温度的要求较高。
发明内容
为了克服现有技术的缺陷，本发明的目的是提供一种低温适应性好的脲基甲酸水解酶，并相应开发一种更高效、简便的制备脲基甲酸水解酶的方法。
本发明提供一种脲基甲酸水解酶的氨基酸序列，所述脲基甲酸水解酶的氨基酸序列如SEQ ID NO.1所示。
本发明提供一种脲基甲酸水解酶基因的核苷酸序列，所述脲基甲酸水解酶的核苷酸序列如SEQ ID NO.2所示。
相应地，本发明提供包含上述脲基甲酸水解酶基因的重组载体、重组菌株以及表达上述脲基甲酸水解酶的方法。本发明提供包含上述脲基甲酸水解酶基因的重组载体，优选为pET21a；本发明提供包含上述脲基甲酸水解酶基因PCR扩增的引物序列，正向引物如SEQ ID NO.3所示，反向引物如SEQ ID NO.4；将本发明的脲基甲酸水解酶基因插入到表达载体合适的限制性酶切位点之间，使其核苷酸序列可操作的与表达调控序列相连接，作为本发明的一个优选的实施方案，具体的是将本发明的脲基甲酸水解酶基因插入到质粒pET21a上的EcoRI和NdeI限制性酶切位点之间，使该核苷酸序列位于启动子下游并受其调控，得到重组大肠杆菌BL21表达质粒pET21a-APH；本发明还提供了一种表达上述脲基甲酸水解酶的方法，包括以下步骤：
1)将氨基酸序列如SEQ ID NO.1所示的脲基甲酸水解酶的编码基因插入到质粒pET21a上的EcoRI和NdeI限制性酶切位点之间，得到重组大肠杆菌BL21表达质粒pET21a-APH；
2)将上述重组载体转化大肠杆菌BL21感受态，得到重组菌株Ecoli BL21-pET21a-APH；
3)将Ecoli BL21-pET21a-APH扩大培养，诱导表达脲基甲酸水解酶，收集菌体；
4)菌体破碎后，通过Ni-NTA亲和层析色谱柱纯化后得到纯化的脲基甲酸水解酶。
相应地，本发明提供上述脲基甲酸水解酶鼠来源的多克隆抗体的制备和检测方法，包括以下步骤：
1)纯化后的脲基甲酸水解酶注射小鼠，采集血清；
2)用步骤1)中得到的血清通过ELISA进行抗体对抗原的效价；
3)步骤2)中的测得的效价符合条件，即进行抗体纯化；
4)将步骤3)中纯化得到的抗体与脲基甲酸水解酶的原始菌株的破碎液进行Western Blotting检测。
本发明的有益效果在于：1)本发明通过PCR技术在喜冷杆菌属中扩增得到脲基甲酸水解酶基因，并且通过NCBI Blast碱基序列比对证明该脲基甲酸水解酶基因碱基序列属于喜冷杆菌属的新的脲基甲酸水解酶基因序列；2)通过基因工程技术得到表达脲基甲酸水解酶蛋白的Ecoli BL21-pET21a-APH重组菌株，诱导表达重组菌株得到脲基甲酸水解酶蛋白；3)利用Ecoli BL21-pET21a-APH重组菌株诱导表达脲基甲酸水解酶蛋白的方法简单，易操作，而且发现了新的脲基甲酸水解酶基因序列，该脲基甲酸水解酶在低温条件下依然具备较强的酶学活性；4)本发明提供了制备和检测该蛋白的多克隆抗体，为进一步开展脲基甲酸水解酶功能分析奠定基础，并为Cryobacterium属的菌种中的鉴定提供证据及低温适应机制的研究提供新的方向。
附图说明
图1为PCR扩增脲基甲酸水解酶基因的琼脂糖凝胶电泳鉴定图；其中M泳道为DNAmarker，1号泳道为PCR扩增脲基甲酸水解酶基因的产物；
图2为表达载体pET21a-APH的构建示意图；
图3为重组菌株Ecoli BL21-pET21a-APH表达脲基甲酸水解酶蛋白的聚丙烯酰胺凝胶电泳鉴定图；其中M泳道为protein marker；1号泳道为脲基甲酸水解酶蛋白；
图4为细菌脲基甲酸水解酶多克隆抗体的Western Blotting分析图。
具体实施方式
以下通过实施例对本发明作进一步的阐述，但不限制本发明。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。本具体实施方式仅为最佳例举，并非对本发明技术方案的限制性实施。
实施例1脲基甲酸水解酶基因的获取
1)获取含有目的基因的菌株
本发明利用喜冷杆菌属菌株，喜冷杆菌属菌株筛选自长白山土壤，具体的筛选过程如专利号为201710034491.5的中国发明专利，该菌株的命名为：Cryobacteriumbaishanse 02，保存于中国典型培养物保藏中心，保藏编号CCTCC NO：M2016604。保藏日期为2016年10月31日，保藏地址为，中国武汉，武汉大学。
2)基因组的提取
利用DNA提取试剂盒(TAKARA Dalian)提取Cryobacteriumbaishanse 02菌株的基因组作为模板，提取的基因组样品于-20℃保存待用。
3)PCR扩增目的基因
设计引物：正向引物如SEQ ID NO.3所示，反向引物如SEQ IDNO.4所示；PCR反应体系如下表1所示：
表1
| 成分 | 体积 |
| 10×PCR缓冲液 | 5μl |
| 引物F | 1μl |
| 引物R | 1μl |
| 模板 | 80 ng |
| MgSO<sub>4</sub> | 2μl |
| dNTP | 5μl |
| 去离子水 | 35μl |
| 总计 | 50μl |
PCR反应条件为：95℃预变性5 min；95℃30 s，55℃30s，68℃延伸2.30 min，共计30个循环；68℃延伸10 min；15℃保持10 min。
将PCR扩增产物经1％琼脂糖凝胶电泳，如图1所示，PCR扩增产物的大小与预计的1926 bp接近，将PCR扩增产物切胶回收，送生物公司测序以准确判断PCR扩增产物的碱基序列，测序委托铂尚生物技术有限公司完成。
测序结果如SEQ ID NO.2所示，将SEQ ID NO.2所示的碱基序列在NCBI Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)上进行碱基序列比对，比对结果与Cryobacterium arcticum strain PAMC27867的脲基甲酸水解酶相似性为91％，即可判断SEQ ID NO.2所示的碱基序列属于Cryobacterium属的新的脲基甲酸水解酶序列。
实施例2 构建脲基甲酸水解酶的表达载体
将实施例1中得到的PCR扩增产物进行胶回收，用限制性内切酶EcoRI和NdeI对PCR扩增产物进行双酶切反应；同时用限制性内切酶EcoRI和NdeI对载体pET21a(+)进行双酶切反应，然后经高效DNA连接酶High Ligation(TOYOBO)催化连接经双酶切反应后的pET21a(+)和PCR扩增产物；构建得到脲基甲酸水解酶的表达载体pET21a-APH，表达载体pET21a-APH的构建如图2所示。
实施例3 表达和纯化细菌脲基甲酸水解酶
将实施例2得到的表达载体pET21a-APH转化入大肠杆菌BL21的感受态菌体中，大肠杆菌BL21的感受态的制备采用CaCl2法，CaCl2法制备大肠杆菌BL21的感受态为实验室常规实验手段，在此不再赘述。将得到的表达载体pET21a-APH转化入大肠杆菌BL21后得到重组菌株Ecoli BL21-pET21a-APH。将Ecoli BL21-pET21a-APH扩大培养至OD600＝0.6后添加1mM IPTG，18℃延时诱导培养表达脲基甲酸水解酶蛋白，诱导培养结束后收集菌体，依次经菌体破碎和Ni-NTA亲和层析色谱柱纯化后得到纯化后的脲基甲酸水解酶蛋白，如图3所示，得到脲基甲酸水解酶蛋白的蛋白分子量在44.3-66.4之间，与理论预计值66.0KD一致，表明纯化得到的蛋白即为脲基甲酸水解酶蛋白。
实施例4 脲基甲酸水解酶活性的测定
底物为：脲基甲酸钾
条件为：PH为9.5，与脲基甲酸水解酶共孵育30min
检测结果如下表：如以25℃的酶活性为100％
由上表可以看出，来源于耐冷细菌的脲基甲酸水解酶在低温下具有一定酶活，最适温度为42℃，相对于其他细菌来源的脲基甲酸水解酶具有很低的最适温度，在工业生产上具有节约能源的优势。
实施例5 脲基甲酸水解酶鼠来源的多克隆抗体的制备和效价检测
取纯化后的脲基甲酸水解酶，采用皮下注射免疫约5周龄的小鼠，40-60μg每次，2-3周免疫一次，共免疫4次；采血检测，通过ELISA方法确定抗体针对抗原的效价，效价大于1:50000进行最终采血制备抗血清，纯化制备多克隆抗体。
ELISA方法包括以下步骤：
1)将抗原用碳酸钠缓冲液(pH 9.6)稀释到10μg/ml，将抗原置于酶标96孔板，每孔100μl孵育1小时；
2)利用PBS-T将酶标板清洗3次；
3)用5％skim milk封闭酶标板1小时，每孔100μl；
4)利用PBS-T将酶标板清洗3次；
5)稀释的血清(1/5000in PBS-T)孵育抗原1小时；
6)利用PBS-T将酶标板清洗3次；
7)每孔加入100μl HRP标记的羊抗鼠抗体(1/5000in PBS-T)，孵育1小时；
8)利用PBS-T将酶标板清洗3次；
9)分光光度计上测420nm下的OD值；
10)通过SDS-PAGE电泳，考马斯亮蓝染色观察纯化的抗体纯度。
实施例6脲基甲酸水解酶鼠来源的多克隆抗体的纯化
抗体纯化是将抗原蛋白与Ni-NTA偶联制备成抗体纯化层析柱，将所得抗血清与PBS按照1:1比例混合后，缓慢经过层析柱，待抗体与抗原结合后用甘氨酸洗脱缓冲液洗脱，获得纯化抗体，纯化抗体利用PBS过夜透析，次日进行浓度、效价测定。
上述纯化抗体浓度测定方法，包括如下步骤：
1)CBB染色液配制：根据样品数量，将5×CBB染色液稀释，充分混匀。
2)根据需要取适量BSA标准蛋白，配制终浓度分别为1mg/ml、0.75mg/ml、0.5mg/ml、0.25mg/ml和0.125mg/ml标准样，并混匀。标准样采用PBS为溶剂。
3)标准曲线绘制：取一块酶标板，按下表格加入试剂：
| 孔号 | A | B | C | D | E | F |
| 标准样浓度(mg/ml) | 0 | 0.125 | 0.25 | 0.5 | 0.75 | 1 |
| 各浓度标准样(μl) | 1 | 1 | 1 | 1 | 1 | 1 |
| 去离子水 | 9 | 9 | 9 | 9 | 9 | 9 |
| CBB染色液 | 200 | 200 | 200 | 200 | 200 | 200 |
| 对应蛋白含量(μg) | 0 | 0.125 | 0.25 | 0.5 | 0.75 | 1 |
| 最终体积(μl) | 210 | 210 | 210 | 210 | 210 | 210 |
振荡混匀后，室温下静置30min；
用酶标仪测定562nm处的吸光值，以不含BSA的光吸收为空白对照；
以蛋白含量(μg)为横坐标，吸收值为纵坐标，绘出标准曲线。
4)样品测定：将待测蛋白样品用去离子水稀释不同浓度梯度，各取1μl并稀释至10μl，再加入200μl 1×CBB，混匀后室温下静置30min，然后以不含蛋白的溶剂为空白对照，测定样品吸收值。
5)根据测得的吸收值，在标准曲线上计算样品的蛋白含量。
计算蛋白浓度：以查得的蛋白含量，并根据稀释倍数计算样品的实际浓度。
实施例6 脲基甲酸水解酶鼠来源的多克隆抗体的Western Blotting检测
1)细菌Cryobacterium baishanse 02培养至OD至约为2.0，取1.5ml离心收集菌体，300μl Tris-HCl重悬，超声破碎取20μl破碎夜与5μl5x SDS上样缓冲液混合，其中10μl进行SDS-PAGE电泳。Magic上样量为0.8μl；
2)上样后，电泳仪恒定为18mA，直到电泳结束；
3)电泳后，将凝胶上蛋白样品转移至PVDF膜，恒定电流为0.23A进行转膜，约为35分钟；
4)电泳结束后将PVDF膜干燥，后于5％skim milk中封闭1小时。
5)PBST清洗5次；
6)用PBST稀释纯化的抗体(1:5000)，孵育1小时；
7)PBST清洗5次；
8)用PBST稀释二抗(1:20000，羊抗鼠-HRP)，孵育1小时；
9)PBST清洗5次，ECL显影，暗室成像。
序列表
<110> 湖北工业大学
<120> 一种脲基甲酸水解酶及其制备方法
<130> PatentIn version 3.5
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Leu Ala Pro Leu Trp Arg Ser Ala Phe Asp Ala His Leu Ala Arg Leu
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Arg Asp Ala Gly Val Thr Leu Val Glu Met Asp Ile Ser Pro Phe Leu
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Glu Ala Ala Ala Leu Leu Tyr Asn Gly Ala Phe Val Ala Glu Arg Tyr
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Ala Ala Val Gly Ala Ala Ile Asp Ser Ala Pro Ala Gly Leu Asp Pro
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Val Val Ala Gly Ile Val Thr Ala Ala Gly His Leu Pro Ala His Arg
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Ala
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tgcgtcaccg cgttcgcccg cgacctcgac accgccgagt cggtgctgag ggtgatggcc 660
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ccggcggccc ccgtgatcgc cgtgccccgc cccgacgacc tcaccgcgct cgcgccgctc 780
tggcggagcg ccttcgacgc ccacctggcc cggttgcgtg acgccggcgt caccctggta 840
gagatggaca tctccccgtt cctcgaggcc gccgcgttgc tgtacaacgg cgccttcgtg 900
gccgagcgtt acgccgccgt cggtgcggcg atcgattcag cacctgcggg gctcgaccct 960
gtcgttgccg gcatcgtcac cgccgccggg cacctgcccg cgcaccggta cgcggccgac 1020
ctggtgcgct tggccgagct gaagctcgcc ggggaactcg ccttcgccgg cgccgacgcc 1080
atgctgctgc ccaccaccac gcaccacccc accctggccg aggtggcggc ggagcccatc 1140
gccgtgaact cccggctagg caccttcacc aacttcgcca atctgttcgg ctaccccgcg 1200
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cgccccttcg ccgacgccgt cgcccgtgac gtggccgccc tgctgctgcg tgtcgaatcg 1320
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ggtgcgcacc tgtccgggct gccgctgcac gaccggatgc gtcggcacgg cgcactgttc 1620
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cggcccggca tgatccgcac gcccgactcc gactccagcg tgctcggcga actgtggcgg 1740
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Claims (6)
1.一种脲基甲酸水解酶，其特征在于：其氨基酸序列如SEQ ID NO.1所示。
2.一种脲基甲酸水解酶基因，其特征在于：编码权利要求1所述的脲基甲酸水解酶；其核苷酸序列如SEQ ID NO.2所示。
3.包含权利要求2所述脲基甲酸水解酶基因的重组载体，其特征在于：所述载体的构建方法为：将氨基酸序列如SEQ ID NO.1所示的脲基甲酸水解酶的编码基因插入到质粒pET21a上的EcoRI和NdeI限制性酶切位点之间，使该核苷酸序列位于启动子下游并受其调控，得到重组大肠杆菌BL21表达质粒pET21a-APH。
4.PCR扩增权利要求2所述脲基甲酸水解酶基因的引物序列，其特征在于：所述引物序列的正向引物如SEQ ID NO.3所示，反向引物如SEQ ID NO.4。
5.一种制备权利要求1所述的脲基甲酸水解酶的方法，其特征在于：包括以下步骤：
1)将氨基酸序列如SEQ ID NO.1所示的脲基甲酸水解酶的编码基因插入到质粒pET21a上的EcoRI和NdeI限制性酶切位点之间，得到重组大肠杆菌BL21表达质粒pET21a-APH；
2)将上述重组载体转化大肠杆菌BL21感受态，得到重组菌株Ecoli BL21-pET21a-APH；
3)将Ecoli BL21-pET21a-APH扩大培养，诱导表达脲基甲酸水解酶，收集菌体；
4)菌体破碎后，通过Ni-NTA亲和层析色谱柱纯化后得到纯化的脲基甲酸水解酶。
6.一种制备和检测权利要求1所述的脲基甲酸水解酶鼠来源的多克隆抗体的方法，其特征在于：包括以下步骤：
1)纯化后的脲基甲酸水解酶注射小鼠，采集血清；
2)用步骤1)中得到的血清通过ELISA进行效价检测；
3)步骤2)中的测得的效价符合条件，即进行抗体纯化；
4)将步骤3)中纯化得到的抗体与脲基甲酸水解酶的原始菌株的破碎液进行WesternBlotting检测。
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