CN107987137B - 一种abc转运蛋白及其制备方法 - Google Patents
一种abc转运蛋白及其制备方法 Download PDFInfo
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- CN107987137B CN107987137B CN201711374880.9A CN201711374880A CN107987137B CN 107987137 B CN107987137 B CN 107987137B CN 201711374880 A CN201711374880 A CN 201711374880A CN 107987137 B CN107987137 B CN 107987137B
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- abc transporter
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Abstract
本发明涉及分子生物学领域，尤其涉及一种ABC转运蛋白及其制备方法。包括以下步骤：1)利用PCR技术扩增得到ABC转运蛋白的克隆，2)构建ABC转运蛋白的表达载体；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ABC转运蛋白。该方法简单，易操作，且发现了新的基因序列的ABC转运蛋白。另外，本发明还提供该ABC转运蛋白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ABC转运蛋白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ABC转运蛋白及其应用。
背景技术
ABC转运蛋白，英文名ATP-binding Cassette(ABC)transporter，是一类ATP驱动泵，对于细胞跨膜转运十分重要。该蛋白对于底物或底物的基团具有特异性。
正常生理条件下，ABC转运蛋白是细菌质膜上糖，氨基酸，磷脂和肽的转运蛋白，是哺乳动物细胞质膜上磷脂、亲脂性药物、胆固醇和其他小分子的转运蛋白。其在肝、小肠和肾等器官细胞质膜分布丰富，能将天然毒物和代谢废物排除体外。
由于有些ABC转运蛋白能够将抗生素或其他抗癌药物泵出细胞而赋予细胞抗药性，近些年来，ABC转运蛋白在医学领域引起了极大关注。事实上，真核细胞最早鉴定的ABC转运蛋白就是从肿瘤细胞和抗药物培养细胞中发现的。
另外，将ABC转运蛋白基因转到具有降解能力的微生物中，可以使工程化后的微生物对污染的环境进行微生物修复，但这一应用在温度较低的地区很难发挥作用。
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ABC转运蛋白，并相应开发一种更高效、简便的制备ABC转运蛋白的方法。
本发明提供一种ABC蛋白的氨基酸序列，所述ABC蛋白的氨基酸序列如SEQ IDNO.1所示。
本发明提供一种ABC蛋白基因的核苷酸序列，所述ABC蛋白的核苷酸序列如SEQ IDNO.2所示。
相应地，本发明提供包含上述ABC蛋白基因的重组载体、重组菌株以及表达上述ABC蛋白的方法。本发明提供包含上述ABC蛋白基因的重组载体，优选为pET21a；本发明提供包含上述ABC蛋白基因PCR扩增的引物序列，正向引物如SEQ ID NO.3所示，反向引物如SEQID NO.4；将本发明的ABC蛋白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ABC蛋白基因插入到质粒pET21a上的EcoRI和XhoI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-ABC；本发明还提供了一种表达上述ABC转运蛋白的方法，包括以下步骤：
1)将氨基酸序列如SEQ ID NO.1所示的ABC转运蛋白的编码基因插入到质粒pET21a上的EcoRI和XhoI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-ABC；
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-ABC；
3)将Ecoli BL21-pET21a-ABC扩大培养，诱导表达ABC转运蛋白，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ABC转运蛋白。
相应地，本发明提供上述ABC转运蛋白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ABC转运蛋白注射小鼠，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ABC转运蛋白的原始菌株的破碎液进行WesternBlotting检测。
本发明的有益效果是：1)本发明通过PCR技术在喜冷杆菌属中扩增得到ABC转运蛋白基因，并且通过NCBI Blast碱基序列比对证明该ABC转运蛋白基因碱基序列属于喜冷杆菌属的新的ABC转运蛋白基因序列；2)通过基因工程技术得到表达ABC转运蛋白的EcoliBL21-pET21a-ABC重组菌株，诱导表达重组菌株得到ABC转运蛋白；3)利用Ecoli BL21-pET21a-ABC重组菌株诱导表达ABC转运蛋白的方法简单，易操作，而且发现了新的ABC转运蛋白基因序列；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ABC转运蛋白功能分析奠定基础，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ABC转运蛋白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扩增天冬氨酸转氨酶基因的产物；
图2为表达载体pET21a-ABC的构建示意图；
图3为重组菌株Ecoli BL21-pET21a-ABC表达ABC转运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ABC转运蛋白。
图4为细菌ABC转运蛋白多克隆抗体的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1ABC转运蛋白基因的获取
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。
2)基因组的提取
利用DNA提取试剂盒(TAKARA Dalian)提取Cryobacterium baishanse 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 ID NO.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 | 80ng |
| 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5min；95℃30s，55℃30s，68℃延伸20min，共计30个循环；68℃延伸10min；15℃保持10min。
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1140bp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扩增产物的核苷酸序列，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所示的核苷酸序列在NCBI Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)上进行核苷酸序列比对，比对结果与Cryobacterium arcticum strain PAMC27867的ABC转运蛋白相似性为97％，与Cryobacterium sp.LW097的ABC转运蛋白相似度为82％，即可判断SEQ ID NO.2所示的核苷酸序列属于Cryobacterium属的新的ABC转运蛋白编码基因序列。
实施例2构建ABC转运蛋白的表达载体
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XhoI对PCR扩增产物进行双酶切反应；同时用限制性内切酶EcoRI和XhoI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ABC转运蛋白的表达载体pET21a-ABC，表达载体pET21a-ABC的构建如图2所示。
实施例3表达和纯化细菌ABC转运蛋白
将实施例2得到的表达载体pET21a-ABC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-ABC转化入大肠杆菌BL21后得到重组菌株Ecoli BL21-pET21a-ABC。将Ecoli BL21-pET21a-ABC扩大培养至OD600＝0.6后添加1mM IPTG，18℃诱导培养表达ABC转运蛋白，待OD约2.5时离心收集菌体，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ABC转运蛋白，如图3所示，得到ABC转运蛋白的蛋白分子量在44.3～66.4之间，与理论预计值56.6KD一致，表明纯化得到的蛋白即为ABC转运蛋白。
实施例4ABC转运蛋白鼠来源的多克隆抗体的制备和效价检测
取纯化后的ABC转运蛋白，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5ABC转运蛋白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6ABC转运蛋白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ABC转运蛋白及其制备方法
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Claims (2)
1.一种ABC转运蛋白的编码基因，其特征在于：其核苷酸序列如SEQ ID NO.2所示。
2.一种制备ABC转运蛋白的方法，其特征在于：包括以下步骤：
1）将核苷酸序列如SEQ ID NO.2 所示的ABC转运蛋白的编码基因插入到质粒 pET21a上的EcoRI和XhoI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-ABC；
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-ABC；
3）将Ecoli BL21-pET21a-ABC扩大培养，诱导表达ABC转运蛋白，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ABC转运蛋白。
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| WO1999031133A1 (en) * | 1997-12-18 | 1999-06-24 | Smithkline Beecham Corporation | Novel abc transporter |
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