CN112125977B - 一种tweak-mv1偶联蛋白及其抗银屑病的应用 - Google Patents
一种tweak-mv1偶联蛋白及其抗银屑病的应用 Download PDFInfo
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Abstract
本发明公开了一种TWEAK‑MV1偶联蛋白及其抗银屑病的应用，属于生物医药技术领域。本发明采用原核表达系统，成功纯化了重组TWEAK蛋白，并将其与MV1蛋白偶联成功，制备出可诱导角质形成细胞凋亡的TWEAK‑MV1蛋白，为银屑病的治疗提供新的选择。
Description
技术领域
本发明属于生物医药领域，涉及一种TWEAK-MV1偶联蛋白及其抗银屑病的应用。
背景技术
银屑病是一种常见慢性炎症性皮肤病，病程长，易复发。目前认为以角质形成细胞为中心的炎症因子释放与免疫调控处于银屑病发病过程的关键地位，其导致角质形成细胞的周期过程异常(增殖活跃，凋亡受抑)。TWEAK一种TNF家族配体新成员，通过与分布于靶细胞膜表面的唯一受体Fn14发生特异性结合而发挥生物学效应。研究报道银屑病皮损部位Fn14显著高表达。传统观点认为，TWEAK/Fn14信号促进体外正常角质形成细胞的凋亡与坏死。但我们对不同炎症微环境下的角质形成细胞进行研究，发现TWEAK刺激具有双向调控细胞命运的作用。利用多种细胞因子模拟体外银屑病炎症微环境，也能促进Fn14高表达，施加TWEAK刺激后诱导角质形成细胞增殖而非凋亡。
TNF-导通过与两种亚类受体(TNFR1与TNFR2)结合，而调控细胞周期变化。其中，TNF-而与TNFR1结合可以导致caspase-8途径激活，引起细胞凋亡；而TNFR2可以拮抗TNFR1引起的caspase激活效应，反而促进NF-应，信号反应及细胞增殖。TNFR1与TNFR2都可以表达于角质形成细胞、血管内皮细胞等，但分布存在偏向性差异，这可能导致对TNF-达刺激的不同反应效果。TWEAK还促进银屑病炎症下角质形成细胞产生C-myc、cIAP2、cFLIP等抗癌蛋白，促使细胞呈现以TNFR2为主的TNF受体分布特征。因此，炎症微环境通过改变TNFR表达谱来影响TWEAK/Fn14信号极性方向，继而调控角质形成细胞增殖与银屑病炎症反应。
cIAP1在细胞质中泛素化受体相互作用的丝氨酸/苏氨酸蛋白激酶1(RIP1)，表皮中的cIAP1缺失导致角质形成细胞死亡。在过表达TNFR2的角质形成细胞中这些细胞中，TWEAK诱导更多的cIAP1向细胞质中分布，且cIAP1的细胞质/核比率随着TWEAK的刺激而增加。然而，通过转染Fn14 siRNA可消除这种作用。通过表面等离子共振测定Fn14，TRAF2，cIAP1和TNFR1(或TNFR2)分子之间的结合亲和力，我们发现Fn14特异性结合TRAF2，其进一步对TNFR1和TNFR2具有结合亲和力；而TRAF2可以特异性结合cIAP1，形成TRAF2-cIAP1复合物。因此，TRAF2是形成Fn14/TRAF2/cIAP1/TNFR复合物的关键分子，是介导TWEAK促进银屑病角质形成细胞增殖的关键节点。
鉴于TWEAK/Fn14通路在银屑病发病过程中的关键作用，如果设计出能够与Fn14特异性识别的、靶向性的抗Fn14单抗/TWEAK-毒素偶合物对银屑病的治疗将具有非常重要的意义。
发明内容
本发明的目的在于提供一种TWEAK-MV1偶联蛋白及其抗银屑病的应用。
为了达到上述目的，本发明采用以下技术方案予以实现：
本发明公开了一种TWEAK-MV1偶联蛋白，该TWEAK-MV1偶联蛋白的氨基酸序列如SED.ID.NO.1所示。
进一步地，编码所述TWEAK-MV1偶联蛋白的核苷酸序列如SED.ID.NO.2所示。
本发明还公开了上述所述的TWEAK-MV1偶联蛋白在制备治疗抗银屑病的药物中的应用。
优选地，所述的药物为诱导角质形成细胞凋亡的药物。
本发明还公开了一种治疗银屑病的药物，该药物由上述的TWEAK-MV1偶联蛋白添加药学上可接受的药用辅料制成。
与现有技术相比，本发明具有以下有益效果：
本发明采用原核表达系统，成功纯化了重组TWEAK蛋白，并将其与MV1蛋白偶联成功，制备出可诱导角质形成细胞凋亡的TWEAK-MV1蛋白，MV1是特异性cIAP1拮抗剂，其可以通过OH→NH替换方式与生物素形成稳定连接并广泛发挥拮抗效应。MV1可以通过诱导cIAP1构象变化而促使自动泛素化，最终发挥TRAF2/cIAP1信号抑制效应。而TWEAK与Fn14的特异性结合，将使TWEAK-MV1耦合物特异性识别高表达Fn14的靶细胞如角质形成细胞，选择性阻断TRAF2/cIAP级联信号，削弱其对角质形成细胞的促增殖作用而导致靶细胞死亡，本发明能够为银屑病的治疗提供新的选择。
附图说明
图1为本发明重组质粒结构图；
图2为本发明的载体信息图；
图3为本发明的重组质粒酶切图；
图4为本发明的融合蛋白表达小试SDS-PAGE分析图；
图5为本发明的融合蛋白镍琼脂糖亲和层析纯化SDS-PAGE分析图；
图6为最终纯化蛋白SDS-PAGE分析图；
图7为TWEAK蛋白浓度测定结果；
图8为TWEAK蛋白飞行时间质谱图；
图9为TWEAK-MV1飞行时间质谱图；
图10为MV1小分子HPLC检测结果图；
图11为TWEAK蛋白HPLC检测结果图；
图12为TWEAK与MV1偶联后的HPLC检测结果图；
图13为银屑病微环境下TWEAK-MV1促进角质形成细胞凋亡结果；其中，A图显示采用质粒成功构建了可溶性TWEAK(sTWEAK)；B图显示将TWEAK-MV1与Fn14阳性角质形成细胞孵育；C图显示将两者都施加于Fn14阳性角质形成细胞，仅TWEAK-MV1明显诱导细胞凋亡。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述：
本项目对TWEAK蛋白序列的亲疏水性、信号肽、跨膜域、基本结构等进行了理论评估，根据评估结果设计质粒构建方案，采用全基因合成的方式构建质粒并亚克隆到pET28a表达载体上，进一步转化到Rosetta(DE3)大肠杆菌感受态细胞，培养，诱导表达，收集菌体，纯化蛋白，最后通过验证获得10mg、纯度>90％的重组TWEAK蛋白，并将MV1与TWEAK蛋白偶联。
1、材料准备
表1
2、TWEAK蛋白原核表达纯化：
2.1质粒构建及验证
扩增目的基因片段：根据基因序列及构建方案设计引物，通过PCR扩增出足够量的PCR产物。
酶切：根据引物设计的酶切位点，分别对PCR产物和质粒载体进行酶切。
目的片段及载体连接：通过连接酶将酶切后的PCR产物和质粒载体进行连接。
获得重组质粒：连接液转入TOP10感受态中，检测筛选出阳性克隆进行测序验证。
2.2蛋白表达
转化：将重组质粒转入大肠杆菌感受态细胞，42℃热激90s后涂布在含有对应卡那霉素和氯霉素的平板上，培养；
活化：挑取单克隆到含有卡那霉素和氯霉素的液体培养基中培养；
诱导：当OD值达到0.6时，添加诱导剂IPTG，继续培养，分别于20℃条件下培养过夜、37℃条件下培养过夜，未添加诱导剂的为阴性对照；
收集菌体：离心，弃上清，收集菌体；
表达检测：在收集到的菌体中加入缓冲液A悬浮，使用超声破碎仪使其充分溶解，离心，离心后的沉淀使用缓冲液B进行溶解，分别对上清和沉淀处理，制样，准备SDS-PAGE检测。
确定条件表达：在含对应卡那霉素和氯霉素的培养基中培养菌液，当OD值达到0.6时，添加0.5mM诱导剂IPTG，37℃条件下培养过夜进行大量表达，离心收集细胞菌体。
2.3蛋白纯化
收集粗蛋白：细胞菌体用缓冲液C溶解、超声破碎，离心收集上清粗蛋白。
平衡：取5ml Ni-NTA，用5倍柱床体积的Binding buffer清洗平衡柱子，流速5ml/min；
孵育：将粗蛋白与平衡后的柱填料孵育1h；
上柱：将孵育后的产物上柱，收集流出；
平衡：用Binding buffer清洗平衡柱子；
洗杂：用Washing buffer洗柱子，并收集流出；
洗脱：用Elution buffer洗脱，收集流出；
纯化检测：对粗蛋白、洗杂流出、洗脱流出分别处理，制样，准备SDS-PAGE检测。
透析浓缩：将纯度较好的组分5透析到50mM Tris，300mM NaCl，10％Glycerol，0.1％SKL，pH 7.5中，透析结束后用PEG20000浓缩，0.22μm滤膜过滤后分装1ml/tube，-80℃保存。
2.4目的蛋白检测
SDS-PAGE检测：对纯化后蛋白进行处理，制样，跑胶，检测分子量。
3、TWEAK-MV1蛋白偶联：
依据MV1结构式，尾端OH键易被NH替换，从而与携带NH键端的分子形成稳定连接。既往报道显示，采用HATU(C10H15F6N6OP)作为激活剂，MV1与二氨基己烷(C6H16N2)在二甲基甲酰胺(DMF)溶液中结合并增加四碳-NH2端尾链；以二异丙基乙胺(C8H19N)、4-AMP(C11H22N2O2)作为反应基质，促使MV1与生物素稳定连接，两者间以四碳-NH为连接桥，即可将MV1连接至TWEAK分子的NH键端。
4、结果
4.1序列信息
4.1.1已优化基因序列
CCATGGGCCATCATCATCATCATCACCGTGCTATCGCAGCACACTACGAGGTCCACCCTCGTCCAGGTCAAGACGGTGCACAGGCTGGTGTAGATGGTACTGTAAGCGGTTGGGAGGAAACCAAGATCAACAGCTCCAGCCCGCTGCGTTACGACCGTCAGATCGGTGAATTCACGGTGATTCGCGCCGGTCTGTACTACCTGTACTGCCAGGTGCACTTCGACGAAGGCAAAGCTGTGTATCTGAAACTGGATCTGCTGGTTAATGGCGTTCTGGCCCTGCGCTGCCTGGAAGAATTCTCCGCGACCGCGGCGTCCTCTCCGGGCCCGCAGCTGCGTCTGTGTCAGGTTTCTGGCCTGCTGCCGCTGCGTCCGGGCTCTTCTCTGCGTATTCGCACTCTGCCGTGGGCTCATCTGAAAGCGGCGCCGTTTCTGACCTATTTTGGCCTGTTCCAGGTTCATTAACTCGAG
4.1.2载体信息
结果参见图1和图2。
4.1.3表达氨基酸序列
偶联后的TWEAK-MV1蛋白长度＝153，MW＝17kDa，Predicted pI＝8.50。
序列信息如下：
MGHHHHHHRAIAAHYEVHPRPGQDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQVHFDEGKAVYLKLDLLVNGVLALRCLEEFSATAASSPGPQLRLCQVSGLLPLRPGSSLRIRTLPWAHLKAAPFLTYFGLFQVH
4.2重组质粒酶切检测
重组质粒的酶切检测结果如图3所示，图中Marker选择SM0331(Thermo)，从图3中琼脂糖凝胶电泳结果可以看出PCR产物经连接酶酶切后的TWEAK目的基因片段与质粒成功融合后的重组质粒。
4.3蛋白表达检测
蛋白表达检测结果参见图4，图中M表示Protein Marker(Cat.No.:C600525)；1表示诱导前总蛋白；2为20℃上清；3为20℃沉淀；4为37℃上清；5为37℃沉淀。通过融合蛋白表达小试SDS-PAGE分析结果，可以看出转入重组质粒后的大肠杆菌感受态细胞超声破碎并离心后，37℃条件下沉淀中有明显的TWEAK蛋白条带。
4.4蛋白纯化检测
蛋白纯化检测结果参见图5，图中M为Protein marker(Cat.No.:C600525)；1为上样；2为流出；3为20mM Imidazole洗脱组分；4为50mM Imidazole洗脱组分；5-6为500mMImidazole洗脱组分；通过融合蛋白镍琼脂糖亲和层析纯化SDS-PAGE，可以在洗脱组分看到明显的TWEAK粗蛋白条带。
4.5融合目的蛋白验证
融合目的蛋白验证结果参见图6，融合蛋白经过纯化，SDS-PAGE电泳分析在理论分子量±5kDa相应位置出现明显条带，可初步确定融合蛋白成功得到了纯化。
4.6浓度测定
采用非干扰型蛋白定量试剂盒Cat.No.:C503071测定蛋白浓度为0.93mg/ml，待测样品体积为10μl，结果图7和下表2所示：
表2
4.7小试偶联结果
TWEAK蛋白和TWEAK-MV1偶联蛋白的飞行时间质谱结果参见图8和图9。
4.8放大偶联结果
MV1小分子HPLC检测结果参见图10和下表3：
表3
TWEAK蛋白HPLC检测结果参见图11和下表4
表4
TWEAK-MV1偶联蛋白的的HPLC检测结果参见图12和下表5：
表5
5、TWEAK-MV1偶联蛋白对银屑病微环境细胞模型的作用
采用M5细胞因子组合(包括IL-1a、IL-17A、IL-22、Oncostatin M和TNF-α)，建立体外银屑病炎症细胞模型。利用M5刺激培养细胞，以模拟银屑病炎症微环境。将细胞培养于6孔板，待接近融合时采用低浓度2％胎牛血清培养细胞48h，然后置换正常培养基并添加M5细胞因子(终浓度10ng/ml，维持2天)。施加外源性TWEAK-MV1、TWEAK或对照物刺激。通过蛋白印迹、荧光等实验，检测细胞周期与生存率变化。结果参见图13，图13中A图显示采用质粒成功构建了可溶性TWEAK(sTWEAK)；B图显示将TWEAK-MV1与Fn14阳性角质形成细胞孵育；C图显示将两者都施加于Fn14阳性角质形成细胞，仅TWEAK-MV1明显诱导细胞凋亡。
结果显示：将TWEAK-MV1与上述角质形成细胞(Fn14阳性)孵育，TWEAK-MV1可结合于细胞表面；将TWEAK、TWEAK-MV1分别施加于这些细胞，仅TWEAK-MV1可明显诱导细胞凋亡，而TWEAK组细胞变化不明显,，表明TWEAK与MV1耦联后诱导角质形成细胞凋亡，未来可为银屑病的治疗提供新的选择。
以上内容仅为说明本发明的技术思想，不能以此限定本发明的保护范围，凡是按照本发明提出的技术思想，在技术方案基础上所做的任何改动，均落入本发明权利要求书的保护范围之内。
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<110> 西安交通大学
<120> 一种TWEAK-MV1偶联蛋白及其抗银屑病的应用
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Claims (3)
1.一种TWEAK-MV1偶联蛋白在制备治疗抗银屑病的药物中的应用，其特征在于，TWEAK-MV1偶联蛋白中TWEAK蛋白的氨基酸序列如SED.ID.NO.1所示，编码TWEAK蛋白的核苷酸序列如SED.ID.NO.2所示。
2.如权利要求1所述的应用，其特征在于，所述的药物为诱导角质形成细胞凋亡的药物。
3.一种治疗银屑病的药物，其特征在于，该药物由TWEAK-MV1偶联蛋白添加药学上可接受的药用辅料制成，TWEAK-MV1偶联蛋白中TWEAK蛋白的氨基酸序列如SED.ID.NO.1所示，编码TWEAK蛋白的核苷酸序列如SED.ID.NO.2所示。
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