CN106399344B - 一种vin-cdtb融合蛋白的制备方法 - Google Patents

一种vin-cdtb融合蛋白的制备方法 Download PDF

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CN106399344B
CN106399344B CN201610402221.0A CN201610402221A CN106399344B CN 106399344 B CN106399344 B CN 106399344B CN 201610402221 A CN201610402221 A CN 201610402221A CN 106399344 B CN106399344 B CN 106399344B
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罗朝领
冯奇
杜庆辉
茅柳娟
马贵芳
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Abstract

本发明公开了一种vin‑cdtb融合蛋白的制备方法，其特征为，通过在大肠杆菌宿主细胞中进行含vin基因和cdtb基因在同一载体中串联表达的载体和工程菌的构建、诱导表达、纯化，制备重组vin‑cdtb融和蛋白。发明还提供了用于检测肠易激综合征(irritable bowel syndrome，IBS)的试剂盒。

Description

一种vin-cdtb融合蛋白的制备方法
技术领域
本发明涉及热肠激综合症诊断领域，具体涉及用于检测热肠激综合症的融合蛋白、制备方法及应用。
背景技术
肠易激综合征与胃肠功能性肠道疾病症状(如腹痛、紧张、紧迫性、不完全撤离，恶心，和腹胀)与肠功能发生变化(如便秘、腹泻，或两者)。肠易激综合症患者也可能经历共病焦虑和抑郁。肠易激综合征是常见的，患病率在3％至28％之间，影响患者的健康和生活质量。肠易激综合症(IBS)是一种常见的胃肠道疾病，其病理生理学不完全知道，虽然它已被证明基因，社会学习因素，饮食，肠微生物群，肠道低年级炎症胃肠道内分泌细胞发挥重大异常的角色。目前的黄金标准是基于症状的诊断标准，开发的1970年代，随着时间的推移和修改。然而，最新发表在2006.3的，罗马III标准，基于症状标准对IBS的诊断还很有限。
研究一直专注于开发新型生物标志物(生理机制、基因、蛋白质或代谢物)帮助肠易激综合症的诊断。循环抗CdtB和抗vinculin抗体生物标记可以作为IBS检测标志物，并为IBS病理生理学提供一些独特的观点。CdtB是由细菌引起急性胃肠炎，感染后的动物模型表明，宿主抗体在宿主肠道与vinculin交叉作用，产生一个IBS类似表型。因此，我们评估循环anti-CdtB和anti-vinculin抗体作为D-IBS在人体的生物标志物。Anti-vinculin、anti-CdtB抗体也出现在感染后肠易激综合症的部分病理生理学中，可以看做D-IBS定向疗法的一个子群。
发明内容
本发明提供用于检测肠易激综合症的融和蛋白，该融合蛋白可与人体血液中相应抗体特异结合，所以可以快速检测热肠激综合症。
本发明另一目的提供上述融和蛋白的制备方法，所述融合蛋白在原核表达系统，表达量高且易于纯化。
本发明另一目的提供上述融和蛋白在制备检测热肠激综合症试剂盒方面的应用，操作简单灵敏度高且特异性强。
本发明的目的采用如下技术方案实现。
通过DNAstar软件对人粘着斑蛋白(vinculin)和空肠弯曲杆菌细胞扩张毒素B(cdtb)进行B细胞抗原表位分析。确定了vinculin 216-918氨基酸序列和cdtb全长序列，通过Iinker把两者直接串联，并命名为vin-cdtb，融和基因序列进行人工合成，
优选的技术方案中，所述编码基因的氨基酸序列如SEQ ID No：4所示，通过引物扩增融和基因序列利用酶切位点插入到pET28a中，并命名为pET28-vin-cdtb，进行测序。
优选的技术方案中，所述融合蛋白的核苷酸序列如SEQ ID No：3所示；转化BL21(DE3)，菌株命名为BL21-VC.诱导表达，超声破碎，取上清进行镍柱亲和纯化，500mM咪唑缓冲液洗脱，获得目的蛋白vin-cdtb。
附图说明
图1是载体构建图
图1中A部分为PET-28a载体图，其中Not1和Nde1为酶切位点；B部分为vin-cdtb目标基因图，其中Not1和Nde1为酶切位点；C部分为插入vin-cdtb目标基因之后的载体图。
图2是vin-cdtb融合蛋白图。
具体实施方式
实施例1基因设计和合成
所述融和蛋白vin-cdtb基因设计如下：通过DNAstar软件对人粘着斑蛋白(vinculin)和空肠弯曲杆菌细胞扩张毒素B(cdtb)进行B细胞抗原表位分析并确定其基因序列，通过Iinker把两者直接串联，设计的vin-cdtb串联融和基因序列进行人工合成。
实施例2融合基因载体构建和诱导表达
融合基因的两侧选择设计PCR引物，并用化学合成法合成两条，分别为：P1：ggaattccat atgaaaaact caaaaaacca aggcatag(5-引物含Nde1限制性酶切位点)，SEQ IDNo：1P2：ataagaatgc ggccgcttaa aattttctaa aatttactgg aaaatgatct g(3-引物含Not1限制性酶切位点)，SEQ ID No：2。以上述合成基因为模板，用引物P1、P2以扩增条件为：94℃；30秒、58℃40秒、72℃2分钟，30个循环PCR扩增vin-cdtb，同时在其两端添加相应的酶切位点。最后连接vin-cdtb融合基因克隆到pET-28a的Nde1和Not1的两个限制性酶切位点之间，重组质粒pET28-vin-cdtb所含基因vin-cdtb序列经测序证实与设计合成的vin-cdtb基因序列一致。提取质粒后再转化入宿主菌BL21(DE3)中，得到重组菌BL21-VC，等到OD600达到0.3和0.6之间时，用终浓度是0.1mM的IPTG进行低温过25摄氏度夜诱导。
实施例3目的蛋白纯化
诱导的含有目的蛋白的细菌被离心收集，超声破菌，离心收集上清。为了提高目的蛋白的纯化纯度，我们对传统的纯化his融合蛋白的方法进行了相关的改进，在诱导表达后的大肠杆菌上清中加入β-巯基乙醇(使终浓度为5mM)。然后利用含300mMNacl，20mM Tris-Hcl和40mM的咪唑缓冲液把目的蛋白结合到HIS Trap FF亲和柱子上，然后用含有300mMNacl，20mM Tris-Hcl和500mM的咪唑缓冲液把目的蛋白洗脱下来。在PBS缓冲液中4℃透析过夜，BCA法测定蛋白浓度为1mg/ml，sds-page分析纯度大于95％。
实施例4利用elisa检测来测定免疫活性
1.包被：用0.05M PH9，碳酸盐包被缓冲液将融和蛋白vin-cdtb稀释至蛋白质含量为1～10μg/ml，在每个聚苯乙烯板的反应孔中加0.1ml，4℃过夜。次日，弃去孔内溶液，用洗涤缓冲液洗3次，每次3分钟。
2.加样：加一定稀释的待检样品0.1ml于上述已包被之反应孔中，置37℃孵育1小时。然后洗涤。(同时做空白孔，阴性对照孔及阳性对照孔)。
3.加酶标抗体：于各反应孔中，加入新鲜稀释的酶标抗体(经滴定后的稀释度)0.1ml，37℃孵育0.5～1小时，洗涤。
4.加底物液显色：于各反应孔中加入临时配制的TMB(四甲基联苯胺)底物溶液0.1ml，37℃10～30分钟。
5.终止反应：于各反应孔中加入2M硫酸0.05ml。
6.结果判定：可于白色背景上，直接用肉眼观察结果：反应孔内颜色越深，阳性程度越强，阴性反应为无色或极浅，依据所呈颜色的深浅，以“+”、“-”号表示。也可测OD值：在ELISA检测仪上，于450nm(若以ABTS显色剂，于410nm)处，以空白对照孔调零后测各孔OD值，若大于规定的阴性对照OD值的2.1倍，即为阳性。
ELISA试剂盒检测结果
Figure GDA0002717983410000041
Figure ISA0000130941470000011
Figure ISA0000130941470000021
Figure ISA0000130941470000031
Figure ISA0000130941470000041
Figure ISA0000130941470000051
Figure ISA0000130941470000061
Figure ISA0000130941470000071

Claims (2)

1.一种vin—cdtb融合蛋白的制备方法，包括如下步骤：1)通过DNAstar软件对人粘着斑蛋白(vinculin)和空肠弯曲杆菌细胞扩张毒素B(cdtb)进行B细胞抗原表位分析，确定了vinculin216—918氨基酸序列和cdtb全长序列，通过linker把两者直接串联，并命名为vin—cdtb，融和基因序列进行人工合成；2)通过引物扩增融和基因序列，利用酶切位点插入到pET28a中，并命名为pET28-vin—cdtb，进行测序；3)转化BL21(DE3)，菌株命名为BL21-VC；4)诱导表达，超声破碎，取上清进行镍柱亲和纯化，500mM咪唑缓冲液洗脱，获得目的蛋白vin—cdtb。
2.按照权利要求1所述方法制备的vin—cdtb融和蛋白在制备检测肠易激综合征试剂盒方面的应用。
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