CN109679963A - 一种重组人视黄醇结合蛋白4N端19-201aa的表达与纯化方法及应用 - Google Patents
一种重组人视黄醇结合蛋白4N端19-201aa的表达与纯化方法及应用 Download PDFInfo
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- CN109679963A CN109679963A CN201910133611.6A CN201910133611A CN109679963A CN 109679963 A CN109679963 A CN 109679963A CN 201910133611 A CN201910133611 A CN 201910133611A CN 109679963 A CN109679963 A CN 109679963A
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Abstract
本发明涉及生物技术领域，具体是一种视黄醇结合蛋白4N端19‑201aa的表达与纯化方法及应用，利用大肠杆菌表达与纯化重组人视黄醇结合蛋白4，提供了重组人的视黄醇结合蛋白4N端19‑201aa表达载体的构建方法，并对所诱导表达出的重组人的视黄醇结合蛋白4N端19‑201aa进行过Ni琼脂糖凝胶层析柱及KCL法切胶回收两步纯化。本发明所述方法制备的重组人的视黄醇结合蛋白4N端19‑201aa具有高产率、高纯度、低成本的特征，提供了一种RBP4蛋白作为诊断试剂盒标准品的技术方案。
Description
技术领域
本发明涉及生物技术领域，具体是一种利用原核表达系统表达重组人视黄醇结合蛋白4N端19-201aa与纯化方法，及其蛋白的应用。
背景技术
视黄醇结合蛋白(retinol-binding protein，RBP)为血中维生素A(VitA)的特异转运蛋白，RBP4是视黄醇类结合蛋白家族中的一员，由184个氨基酸残基和3个二硫键组成。RBP4主要在肝细胞粗面内质网合成，约15％～30％在脂肪组织合成，其广泛分布于血液、脑脊液、尿液及其他体液中。肝脏释放RBP4受血清视黄醇浓度的调节，当血清视黄醇浓度减低时RBP4释放增加，反之则减少。RBP4也像其他脂肪因子一样，参与代谢综合征、胰岛素抵抗、2型糖尿病的发病机制，并与慢性肝病、慢性肾病、心脑血管疾病、肝癌、结直肠癌等有着密切联系，提示该蛋白可能作为候选生物标志物，对与该蛋白相关的疾病诊断可能具有辅助意义。目前检测RBP4水平的方法通常有酶联免疫吸附测定法、沉淀法、化学发光免疫法等，特别是酶联免疫吸附测定法较为常用，且易于检测。因此对于RBP4试剂盒的推广与应用有益于该RBP4蛋白相关的疾病诊断。
发明内容
本发明的目的是提供一种原核表达的高产率、高纯度、低成本的重组人视黄醇结合蛋白4N端19-201aa的蛋白表达与纯化方法，及其作为ELASE检测试剂盒的蛋白标准品的应用。
本发明通过以下技术方案达到上述目的：一种重组人视黄醇结合蛋白4N端19-201aa的基因，所述的RBP4蛋白N端19-201aa的核苷酸序列如序列表SEQ ID NO.1所示。
所述的RBP4蛋白4N端19-201aa的氨基酸序列如序列表SEQ ID NO.2所示。
所述的人RBP4蛋白目的基因序列为RBP4基因第55-603位核苷酸序列，其上游引物55F(B)如序列表SEQ ID NO.3所示：AAAGGATCCGAGCGCGACTGCCGAGTGAG，下游引物603R(H)如序列表SEQ ID NO.4所示：GGGAAGCTTCTACAAAAGGTTTCTTTCTG。
重组人RBP4蛋白表达载体为pET30a(+)-RBP4(55-603bp)。
一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，包括：
获取人的RBP4目的基因序列后连接到pET30a(+)载体多克隆位点上，得到重组人的RBP4蛋白表达载体，该载体含有6X His纯化标签，并将所述的重组表达载体转化至宿主细胞大肠杆菌BL21(DE3)中，得到重组工程菌株；IPTG诱导重组工程菌株表达重组人的RBP4蛋白，分离获得人视黄醇结合蛋白4。
诱导RBP4蛋白表达条件：28℃，200rpm条件下振荡培养约2.5h-3h至对数生长期，加入终浓度为1mM的IPTG诱导4h。
诱导表达的重组人RBP4蛋白的分离纯化方法为：用平衡缓冲液溶解包涵体，离心后收集上清液；将上清液负载到Ni琼脂糖凝胶层析柱中，然后依次用50mM咪唑洗脱液、100mM咪唑洗脱液、150mM咪唑洗脱液、200mM洗脱液、500mM咪唑洗脱液梯度洗脱目的蛋白，收集洗脱液；将洗脱液跑SDS-PAGE电泳后进行250mM KCl染色切胶回收。
所述的平衡缓冲液组成为：20mM Tris-HCI，pH 7.9，500mM NaCl，10mM咪唑，6M尿素。
所述的咪唑洗脱液梯度组成为：
20mM Tris-HCI，pH 7.9，500mM NaCl，50mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，100mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，150mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，200mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，500mM咪唑，6M尿素。
所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法分离获得人视黄醇结合蛋白4在制备试剂盒蛋白标准品中的应用方法，包被RBP4捕获抗体室温过夜，加稀释液封闭1小时，将上述重组人视黄醇结合蛋白4梯度稀释加入室温孵育1小时，加入RBP4检测抗体孵育1小时，加入链霉亲和素-HRP孵育20分钟，加入显色液孵育20分钟，最后加人终止液，测OD值。所述的试剂盒蛋白标准品构建标准曲线的梯度浓度组成及在450nm测的OD值为：
100ng/mL、3.255；50ng/mL、2.346；25ng/mL、1.468；12.5ng/mL、0.948；6.25ng/mL、0.712；3.125ng/mL、0.573；1.5625ng/mL、0.534；0ng/mL(空白孔)、0.461。重组人视黄醇结合蛋白4梯度稀释浓度及OD值构建的标准曲线拟合度高，r在0.99以上，S为0.16，能作为ELISA试剂盒的定量标准品。
本发明突出的优点是:
目的蛋白表达纯化方法简易，成本低，经过Ni琼脂糖凝胶层析柱纯化和切胶二次纯化，最终可以得到高产率、高纯度的重组蛋白，而且重组蛋白能应用于ELASE试剂盒，作为定量的标准品，标准曲线的拟合度高。
附图说明
图1是目的基因扩增图。
其中M：核酸Marker，1：黑色箭头所示位置为目的基因RBP4(55-603bp)。
图2是重组RBP4蛋白N端19-201aa诱导表达电泳图。
其中M：蛋白Marker，kDa:蛋白质分子质量,1：重组蛋白沉淀1mMIPTG诱导，2：重组蛋白沉淀不诱导，3：pET30a(+)对照沉淀1mMIPTG诱导，4：pET30a(+)对照沉淀不诱导，5：重组蛋白上清1mMIPTG诱导，6：重组蛋白上清不诱导，7：pET30a(+)对照上清1mMIPTG诱导，8：pET30a(+)对照上清不诱导。
图3是重组RBP4蛋白N端19-201aa梯度洗脱电泳图。
其中M：蛋白Marker，kDa:蛋白质分子质量,1-2:50mM咪唑洗脱液蛋白，3-4：100mM咪唑洗脱液蛋白，5-6:150mM咪唑洗脱液蛋白，7-8:200mM咪唑洗脱液蛋白，9:500mM咪唑洗脱液蛋白。
图4是重组RBP4蛋白N端19-201aa梯度洗脱后，结合KCL法切胶回收蛋白浓缩液电泳图。
其中M：蛋白Marker，kDa:蛋白质分子质量,1:切胶回收蛋白浓缩液。
图5是重组RBP4蛋白N端19-201aa蛋白标准品，制备的标准曲线。
其中X:重组RBP4蛋白浓度，Y:OD值，r:相关系数，S:标准误差。
具体实施方式
实施例1
表达载体pET30a(+)-RBP4(55-603bp)的构建
1.1PCR获取RBP4(55-603bp)
使用TaKaRa公司RNA提取试剂盒提取7702人肝细胞Total RNA，以反转录得到的cDNA为模板扩增得到目的片段，PCR反应体系：上游、下游引物各0.4ul，模板2.0ul，PCR PfuSuperMix 10ul，ddH2O 7.2ul；95℃预变性1min，95℃变性30s、61℃退火60s、72℃延伸60s、38个循环，72℃延伸5min，PCR产物经1.0％琼脂糖凝胶电泳鉴定。由图1可见，在约600bp处有一清晰的亮带，与预期大小吻合。
上游引物：AAAGGATCCGAGCGCGACTGCCGAGTGAG(其中下划线为BamH I限制性酶切位点)
下游引物：GGGAAGCTTCTACAAAAGGTTTCTTTCTG(其中下划线为Hind III限制性酶切位点)
1.2pET30a(+)-RBP4(55-603bp)表达载体的构建
(1)酶切：上述PCR产物经纯化试剂盒纯化后，采用BamH I、Hind III酶进行pET30a(+)与纯化PCR产物的双酶切，37℃反应4h，应用OMEGA公司的DNA纯化试剂盒进行纯化。
(2)连接：目的片段6u1，质粒2ul，T4Buffer 1ul，T4连接酶1u1，室温连接4h。
(3)转化：将连接产物转化到50微升Stbl3感受态细胞中，冰浴30min，42℃热激45S，冰浴2min，加入800ul的LB培养液，37℃、200rpm振荡预培养1h，最终涂布于含卡纳霉素抗性的LB培养板，37℃培养过夜。
(4)验证：挑取单菌落进行PCR验证，筛选阳性克隆送去测序公司进行测序鉴定，测序正确的重组质粒命名为pET30a(+)-RBP4(55-603bp)。
实施例2
人重组RBP4蛋白N端19-201aa的原核表达
2.1将pET30a(+)-RBP4(55-603bp)重组质粒转化至表达宿主细胞大肠杆菌BL21(DE3)中，得到重组工程菌株，即BL21-pET30a(+)-RBP4(55-603bp)。
2.2诱导表达：挑取重组工程菌株BL21-pET30a(+)-RBP4(19-603bp)接种于10mLLB液体培养基中(含25ug/mL卡纳霉素)，37℃，200rpm过夜。次日取1mL BL21-pET30a(+)-RBP4(19-603bp)菌液至新的10mLLB液体培养基中(含25ug/mL卡纳霉素)，37℃，200rpm条件下振荡培养约3h，加入终浓度为1mM的IPTG诱导，同时进行质粒pET30a(+)阴性对照，28℃，200rpm诱导4h。菌体于4℃，12000g离心1min，收集下层菌体。用适量PBS缓冲液重悬菌体，进行12％常规SDS-PAGE电泳及考马斯亮蓝方法鉴定，结果如图2显示。
实施例3
人重组RBP4蛋白N端19-201aa的纯化
3.1包涵体处理：诱导表达150mL菌体，具体方法如实施例2所述，用适量预冷的PBS缓冲液重悬菌体，冰浴超声破碎(380W，60min，5s开，2s关)后于4℃，12000rpm离心10min，收集沉淀包涵体。用适量洗涤缓冲液(20mM Tris-HCI，pH 7.9，500mM NaCl，10mM咪唑，1M尿素)清洗包涵体，4℃，12000rpm离心10min，反复操作几遍，直至清洗干净包涵体。加平衡缓冲液(20mM Tris-HCI，pH 7.9，500mM NaCl，10mM咪唑，6M尿素)溶解包涵体，冰浴1小时后4℃，10000g离心20min，收集上清液。
3.2过层析柱：将上清蛋白溶液负载到Ni琼脂糖凝胶层析柱中，置于4℃摇床里吸附2小时。使用15倍柱体积的平衡缓冲液(20mM Tris-HCI，pH 7.9，500mM NaCl，10mM咪唑，6M尿素)冲洗柱子，然后依次使用1倍柱体积含50mM咪唑洗脱液(20mM Tris-HCI，pH 7.9，500mM NaCl，50mM咪唑，6M尿素)、100mM咪唑洗脱液(20mM Tris-HCI，pH 7.9，500mM NaCl，200mM咪唑，6M尿素)、150mM咪唑洗脱液(20mM Tris-HCI，pH 7.9，500mM NaCl，150mM咪唑，6M尿素)、200mM咪唑洗脱液(20mM Tris-HCI，pH 7.9，500mM NaCl，200mM咪唑，6M尿素)、500mM咪唑洗脱液(20mM Tris-HCI，pH 7.9，500mM NaCl，500mM咪唑，6M尿素)进行梯度洗脱目的蛋白，收集洗脱液。12％SDS-PAGE电泳及考马斯亮蓝方法鉴定，结果如图3显示。
3.3切胶进一步纯化：将收集好的洗脱液进行12％的SDS-PAGE电泳(采用不插入梳子的方法进行跑胶)，用预冷的250mM KCl染色约3分钟，切下明显的灰白色凝胶条带，将凝胶条浸泡于PBS缓冲液里，直至无色透明，最后把无色透明胶条冰浴捣碎置于预冷的PBS缓冲液中4℃过夜。次日吸取上层液体到新的管子里，PBS反复抽提蛋白，最终使用超滤管(截留量10KDa)于4℃，3500g离心20min进行浓缩、脱盐，得到纯化蛋白浓缩液。将最终得到的蛋白经12％SDS-PAGE电泳及考马斯亮蓝方法鉴定，结果如图4显示，并进行质谱验证该蛋白为人重组RBP4蛋白；经BCA试剂盒测定纯化蛋白浓度后发现150mL菌体可得到4.00mg目的蛋白，最终将纯化蛋白浓缩液进行冻干保存。
实施例4
本实施例为本发明所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法分离获得人视黄醇结合蛋白4在制备试剂盒蛋白标准品中的应用实例，包括如下步骤：
4.1包被：首先将RBP4捕获抗体稀释到工作浓度，室温包被过夜。
4.2封闭：加入300微升1％BSA封闭RBP4捕获抗体，室温孵育1小时。
4.3加入标准品：将重组视黄醇结合蛋白4梯度稀释为：100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL、1.5625ng/mL、0ng/mL(空白孔)，按每孔100微升加入。
4.4加入检测抗体：按每孔100微升RBP4检测抗体加入，室温孵育1小时。
4.5加入链霉亲和素-HRP：按每孔100微升链霉亲和素-HRP加入，室温孵育1小时。
4.6加入显色液：按每孔100微升显色液加入，在避光条件下室温孵育20分钟。
4.7加入终止液：按每孔500微升终止液加入，在避光条件下室温孵育20分钟。
4.8测OD值：使用450nm波长测OD值，建立重组视黄醇结合蛋白4的标准曲线。
4.9定量分析：根据重组视黄醇结合蛋白4制作的标准曲线，定量分析血清样本中RBP4蛋白的浓度。
从图5显示结果看出，重组人视黄醇结合蛋白4梯度稀释浓度及OD值构建的标准曲线拟合度高，r在0.99以上，S为0.16，能作为ELISA试剂盒的定量标准品。
序列表
<110> 广西医科大学
<120> 一种重组人视黄醇结合蛋白4 N端19-201aa的表达与纯化方法及应用
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atcgtcgcgg agttctccgt ggacgagacc ggccagatga gcgccacagc caagggccga 180
gtccgtcttt tgaataactg ggacgtgtgc gcagacatgg tgggcacctt cacagacacc 240
gaggaccctg ccaagttcaa gatgaagtac tggggcgtag cctcctttct ccagaaagga 300
aatgatgacc actggatcgt cgacacagac tacgacacgt atgccgtgca gtactcctgc 360
cgcctcctga acctcgatgg cacctgtgct gacagctact ccttcgtgtt ttcccgggac 420
cccaacggcc tgcccccaga agcgcagaag attgtaaggc agcggcagga ggagctgtgc 480
ctggccaggc agtacaggct gatcgtccac aacggttact gcgatggcag atcagaaaga 540
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Claims (10)
1.一种重组人视黄醇结合蛋白4N端19-201aa的基因，其特征在于，所述的RBP4蛋白N端19-201aa的核苷酸序列如序列表SEQ ID NO.1所示。
2.根据权利要求1所述的一种重组人视黄醇结合蛋白4N端19-201aa，其特征在于，所述的RBP4蛋白4N端19-201aa的氨基酸序列如序列表SEQ ID NO.2所示。
3.根据权利要求1所述的一种重组人视黄醇结合蛋白4N端19-201aa的基因表达方法，其特征在于，所述的人RBP4蛋白目的基因序列为RBP4基因第55-603位核苷酸序列，其上游引物55F(B)如序列表SEQ ID NO.3所示：AAAGGATCCGAGCGCGACTGCCGAGTGAG，下游引物603R(H)如序列表SEQ ID NO.4所示：GGGAAGCTTCTACAAAAGGTTTCTTTCTG。
4.根据权利要求1所述的一种重组人视黄醇结合蛋白4N端19-201aa的基因，其特征在于，重组人RBP4蛋白表达载体为pET30a(+)-RBP4(55-603bp)。
5.一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，其特征在于，包括：
获取人的RBP4目的基因序列后连接到pET30a(+)载体多克隆位点上，得到重组人的RBP4蛋白表达载体，该载体含有6X His纯化标签，并将所述的重组表达载体转化至宿主细胞大肠杆菌BL21(DE3)中，得到重组工程菌株；IPTG诱导重组工程菌株表达重组人的RBP4蛋白，分离获得人视黄醇结合蛋白4。
6.根据权利要求5所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，其特征在于，诱导RBP4蛋白表达条件：28℃，200rpm条件下振荡培养约2.5h-3h至对数生长期，加入终浓度为1mM的IPTG诱导4h。
7.根据权利要求5所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，其特征在于，诱导表达的重组人RBP4蛋白的分离纯化方法为：用平衡缓冲液溶解包涵体，离心后收集上清液；将上清液负载到Ni琼脂糖凝胶层析柱中，然后依次用50mM咪唑洗脱液、100mM咪唑洗脱液、150mM咪唑洗脱液、200mM洗脱液、500mM咪唑洗脱液梯度洗脱目的蛋白，收集洗脱液；将洗脱液跑SDS-PAGE电泳后进行250mM KCl染色切胶回收。
8.根据权利要求7所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，其特征在于，所述的平衡缓冲液组成为：20mM Tris-HCI，pH 7.9，500mM NaCl，10mM咪唑，6M尿素。
9.根据权利要求7所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法，其特征在于，所述的咪唑洗脱液梯度组成为：
20mM Tris-HCI，pH 7.9，500mM NaCl，50mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，100mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，150mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，200mM咪唑，6M尿素；
20mM Tris-HCI，pH 7.9，500mM NaCl，500mM咪唑，6M尿素。
10.根据权利要求5所述的一种重组人视黄醇结合蛋白4N端19-201aa基因的表达方法分离获得人视黄醇结合蛋白4在制备试剂盒蛋白标准品中的应用，其特征在于，包被RBP4捕获抗体室温过夜，加稀释液封闭1小时，将上述重组人视黄醇结合蛋白4梯度稀释加入室温孵育1小时，加入RBP4检测抗体孵育1小时，加入链霉亲和素-HRP孵育20分钟，加入显色液孵育20分钟，最后加人终止液，测OD值，所述的试剂盒蛋白标准品构建标准曲线的梯度浓度组成及在450nm测的OD值为：
100ng/mL、3.255；50ng/mL、2.346；25ng/mL、1.468；12.5ng/mL、0.948；6.25ng/mL、0.712；3.125ng/mL、0.573；1.5625ng/mL、0.534；0ng/mL(空白孔)、0.461，重组人视黄醇结合蛋白4梯度稀释浓度及OD值构建的标准曲线拟合度高，r在0.99以上，S为0.16，能作为ELISA试剂盒的定量标准品。
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Application publication date: 20190426 |