CN106986930B - 一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的蛋白和应用 - Google Patents
一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的蛋白和应用。所述的rhMOG30‑154蛋白，其氨基酸序列如SEQ ID NO.1所示。本发明创新使用人源MOG胞外抗原结构域重组人MOG30‑154(以下简称rhMOG30‑154)皮下多点免疫的方法建立与人类病理特征更为相似的复发缓解型食蟹猴EAE动物模型(食蟹猴实验性自身免疫性脑脊髓炎模型)。
Description
技术领域：
本发明属于医学研究领域，具体涉及一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的蛋白和应用。
背景技术：
多发性硬化(multiple sclerosis,MS)是一种慢性中枢神经系统(centralnervous system,CNS)自身免疫性疾病，表现为局灶性炎性浸润(视神经、脑、脊髓)、脱髓鞘以及轴突损伤、胶质增生等，为临床神经系统的疑难重病，对其病理过程、发病机制的研究以及治疗药物的筛选和评价都需要合适的动物模型。实验性自身免疫性脑脊髓炎模型(experimental autoimmune encephalomyelitis，EAE)是目前常用的多发性硬化疾病动物模型，该模型可发生白质和深灰质脱髓鞘，生化、免疫及病理特征都与MS非常相似，因此被普遍用于MS的致病机制及临床前研究。
模型制备常用的致敏源为脑或脊髓组织匀浆、髓鞘碱性蛋白(myelin basicprotein,MBP)、蛋白脂质蛋白(proteolipid protein,PLP)和髓磷脂少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)，其中MOG是一种表达在少突胶质细胞最外层的跨膜糖蛋白，尽管含量极少，约占髓鞘总蛋白的0.05％，但由于它存在于髓鞘膜和少突胶质细胞的最外层，故具有高度免疫原性，具有较强的致脑炎作用，且MOG是唯一既能引起脱髓鞘抗体反应又能引起T细胞反应的中枢神经系统髓鞘蛋白成分。许多证据证明MOG和抗MOG抗体在MS的发病过程中起重要作用，故近年来国外学者开始应用MOG诱导的EAE作为研究MS的理想模型。
大多药物尤其是生物技术类药物，用在啮齿类动物、兔、猪等动物模型时效果显著，但临床评估却效果不佳甚至产生不利影响，这与药物本身的特性及动物本身与人类的遗传及病生理差异等因素相关。因此，与人类非常近缘的非人灵长类动物成为新药开发和研究人类疾病十分重要的实验动物。根据FDA的规定，所有二类以上新药都必须经灵长类动物实验合格后方可进入临床研究，非人灵长类实验动物是人类的最近属动物，非人灵长类动物MS模型与人类MS无论在病生理表现上，还是在磁共振影像(MR)表现、临床及免疫反应等方面有许多相似的特征，在用于MS研究时有许多其它动物模型不能比拟之处，是研究人类疾病机理及临床药理等的理想动物模型。
发明内容：
本发明的第一个目的是提供一种能够诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的人源MOG胞外抗原结构域重组人MOG30-154。
本发明的人源MOG胞外抗原结构域重组人MOG30-154(以下简称rhMOG30-154)，其氨基酸序列如SEQ ID NO.1所示。
本发明的第二个目的是rhMOG30-154在制备诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的药物中的应用。
本发明的第三个目的是提供一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的药物，其特征在于，其含有rhMOG30-154作为活性成分。
本发明第四个目的是提供一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的方法，其特征在于，是以rhMOG30-154作为抗原，免疫食蟹猴，得到食蟹猴实验性自身免疫性脑脊髓炎动物模型。
本发明创新使用人源MOG胞外抗原结构域重组人MOG30-154(以下简称rhMOG30-154)皮下多点免疫的方法建立与人类病理特征更为相似的复发缓解型食蟹猴EAE动物模型(食蟹猴实验性自身免疫性脑脊髓炎模型)。
本发明结合食蟹猴在我国资源丰富，遗传背景清楚，个体小，繁殖周期短，性格温顺，饲养成本低，易操作，节约供试品等优点，本发明成功制备的食蟹猴EAE动物模型可为人类多发硬化病和脑脊髓炎的药物研发提供技术平台。在造模过程中通过视频软件全程监控动物动态，对各类指标全方位的进行综合评估，找出相对较客观的量化评价标准，成功建立rhMOG30-154诱导EAE模型的标准化评价体系。同时引入核磁共振影像学(MRI)为MS的早期诊断提供帮助，并为MS的机制研究及临床疗效评价提供依据。
附图说明：
图1是pET28a-rhMOG30-154重组质粒构建示意图；
图2是重组质粒双酶切后琼脂糖凝胶电泳检测结果，1为重组质粒；
图3是重组质粒中目的蛋白编码基因测序图；
图4是12％SDS-PAGE电泳显示rhMOG30-154蛋白的诱导表达时间图，1、2、3、4、5、6、7、8、9表示分别诱导0、1、2、3、4、5、6、7、8h；
图5是12％SDS-PAGE电泳显示rhMOG30-154蛋白的表达形式鉴定图；
图6是12％SDS-PAGE电泳显示rhMOG30-154蛋白的诱导、纯化图；
图7是rhMOG30-154乳剂皮下注射食蟹猴的注射部位和免疫效果图；
图8是rhMOG30-154诱导的食蟹猴EAE模型的体重变化与临床评分图；
图9是rhMOG30-154诱导的食蟹猴EAE模型不同临床评分下的的脑部核磁共振成像(MRI)图；
图10是rhMOG30-154诱导的食蟹猴EAE模型的脑和脊髓组织病理切片图(HE染色)。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
1、菌种、质粒和动物：
rhMOG30-154基因由上海捷瑞生物工程有限公司合成并转入pET28a质粒载体；E.coliBL21(DE3)感受态细胞购自天根生化科技(北京)有限公司；3只食蟹猴(Macacafascicularis，雌性，4-5岁，体重3-4kg)，购自广东蓝岛生物技术有限公司。
2、主要试剂：
完全弗氏佐剂(complete Freund's adjuvant,CFA)，购自美国Sigma公司；百日咳毒素(pertussis toxin)购自美国Enzo公司；舒泰(Zoletil 50)购自法国Virbac公司。
3、主要仪器：
海康威视监控器ivms-4200；核磁共振仪(GE)
实施例1：
一、构建重组质粒pET28a-rhMOG30-154及相应重组工程菌
通过NCBI查得hMOG的氨基酸序列(GenBank:AAH35938.1)，其第30-154位氨基酸序列(多肽)命名为rhMOG30-154，具体氨基酸序列如SEQ ID NO.1所示，由上海捷瑞生物工程有限公司合成rhMOG30-154的编码基因，利用pET-28a(+)质粒本身带有的6×His标签，选择合适的酶切位点NcoI和XhoI，经NcoI和XhoI双酶切后插入同样经NcoI和XhoI双酶切的pET28a载体质粒中，构建重组质粒pET28a-rhMOG30-154，具体构建方法如图1所示。
将重组质粒pET28a-rhMOG30-154转化至大肠杆菌DH5a感受态细胞中，涂布到LB固体培养基(含50μg/mL Kan)上，37℃培养12-24h，从平板上挑取单菌落，接种至5mL LB培养基(含50μg/mL Kan)中37℃振荡培养过夜。抽提质粒，双酶切验证，如图2所示，重组质粒pET28a-rhMOG30-154大小为5611bp，用Nco I和Xho I双酶切后，酶切结果见图2泳道1，经双酶切后得到大片段5231bp，位置正确，在对应Marker 500bp下方可看到一条微弱条带，对应双酶切切下的380bp小片段。将验证正确的阳性克隆送上海捷瑞生物工程有限公司进行测序，测序引物为T7启动子和T7终止子通用引物。测序结果如图3所示，经比对后序列完全正确，成功构建了携带有重组质粒pET28a-rhMOG30-154的大肠杆菌BL21表达菌，命名为重组工程菌。
二、目的蛋白诱导时间的测定
将步骤一的重组工程菌接种至5mL LB液体培养基中(含50μg/mL Kan)，37℃振荡培养12h，将其作为种子液以1％接种量转接入新鲜的200mL液体LB培养基中(含50μg/mLKan)，根据生长曲线确定对数生长期在此加入无菌0.5mol/L的乳糖溶液至终浓度为5mmol/L，37℃诱导培养，诱导0、1、2、3、4、5、6、7、8h取样，制样后进行12％SDS-PAGE蛋白电泳分析以确定目的蛋白最高表达时间。如图4所示，诱导7h后表达量最高。
三、重组工程菌诱导表达
从菌落平板上挑取步骤一的重组工程菌单克隆至含有相应Kan抗性的5ml LB培养基中,37℃，220rpm摇床中震荡过夜培养。第二天，将其作为种子液以1％接种量转接入200mL LB液体培养基中(含50μg/mL Kan)，37℃，250rpm条件下培养，当菌体长到对数生长期时(大约4h，OD600nm为0.6左右)，加入无菌0.5mol/L的乳糖溶液至终浓度为5mmol/L进行诱导，继续培养7小时后，4℃，6000rpm离心20分钟取沉淀，收集菌体，每1L培养物湿菌得量为9-12.5g，湿菌体冻存于-20℃待破碎。
四、rhMOG30-154包涵体蛋白的洗涤、变性、纯化及复性方法
按每1g步骤三的湿菌体悬浮于20ml的菌体裂解缓冲液(20mmol/L Tris-HCl缓冲液，5mmol/L EDTA，pH8.0)中，充分混匀，冰上超声破碎(功率900W×60％，超声3s、间歇3s，超声20min)。超声裂解液4℃，12000rpm，离心20min，分别收集上清和沉淀，用12％SDS-PAGE蛋白电泳检测分析，确定融合蛋白的表达形式。如图5所示，条带1、2分别为乳糖诱导0h、7h总蛋白表达情况，可以看出，乳糖诱导目的蛋白大量表达；条带3、4分别为菌体破碎离心后上清、沉淀蛋白表达情况，目标蛋白主要在沉淀中(条带4)，即以包涵体的形式表达。
每克包涵体依次用20ml洗涤液Ⅰ(20mmol/L Tris-HCl缓冲液，pH8.0)、洗涤液Ⅱ(2mol/L的尿素)、洗涤液Ⅲ(1％Triton X-100)洗涤，洗涤后的包涵体沉淀，按每克湿重加入40ml包涵体溶解液(8mol/L尿素，20mmol/L Tris-HCl缓冲液，500mmol/LNacl，5mmol/L咪唑，PH8.0)，4℃搅拌6h以上，沉淀用8M尿素变性处理后目的蛋白大部分重新溶解到上清中(图5条带6)，然后再12000rpm离心20min，弃沉淀，收集上清。将溶解后的目的蛋白上清用0.22μm滤膜过滤，滤液经恒流泵加入Ni-NTA，分别用10mM(图6左条带2)、300mM(图6左条带3)、1M(图6条左带4)的咪唑溶液冲洗，将每个洗脱浓度的洗脱液收集进行蛋白电泳验证，结果如图6左图所示，300mM咪唑可将目的目的蛋白从镍柱上洗脱，且蛋白较纯。收集的含高纯度目的蛋白的上清液在不断搅拌的情况下缓慢滴加到复性缓冲液(50mM Tris-HCl，50mMNaCl，0.5mM EDTA，5％纯甘油，2％(m/v)L-Ariginine，2mM GSH，0.2mM GSSG，PH8.8)中，使蛋白终浓度在0.1-0.2mg/mL之间，于4℃静置24-36h，复性后的蛋白溶液透析除盐后于冻干机中冻干成粉末保存，结果如图6右图所示，条带1为乳糖诱导0h总蛋白表达情况，条带2为乳糖诱导7h总蛋白表达情况，条带3为经镍柱纯化后的目的蛋白，条带4为稀释复性后的目的蛋白，条带5为冻干后的目的蛋白(rhMOG30-154蛋白)，纯度可以达到95.0％以上。
五、rhMOG30-154蛋白诱导复发缓解型食蟹猴EAE模型
将rhMOG30-154蛋白(1mg/ml)与CFA等体积混合，用玻璃注射器抽打至油包水状态，制成抗原乳剂。食蟹猴用舒泰(Zoletil 50)按4-6mg/kg剂量麻醉后，皮下10点注射抗原乳剂，每点注射100μl，6处背部，2处股腹沟，2处腋窝(图7)。免疫当天第0h和48h腹腔注射2μg/ml，2.5ml/只百日咳毒素(pertussis toxin)，自免疫后每日进行临床评分，每周测体重一次。20天内不发病者(score<2)于致敏后第21天相同剂量皮下追加免疫。在发病初期及中期，通过MRI影像学监测脑部病变大小及空间分布。
实验结果：2只食蟹猴在免疫两次后表现出明显MS(多发性硬化)临床症状，1只食蟹猴在免疫3次后表现出明显MS临床症状，且病情均表现为为复发缓解型，如图8所示为1只猴的临床病程图和体重变化趋势图。显示病情为复发缓解型，体重变化与病情密切相关。图9是不同临床评分下的脑部不同轴向核磁共振成像(MRI)图，可见临床评分与脑部病灶的关系。hMOG30-154诱导的食蟹猴EAE模型的脑和脊髓组织病理切片图(HE染色)如图10所示，大脑、小脑、脑干部位均有大量炎性细胞浸润，大脑、脑干部位已形成广泛的血管套，并延伸入临近实质区域，脊髓有少量炎性细胞浸润。
序列表
<110> 广东省生物资源应用研究所
<120> 一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的蛋白和应用
<160> 1
<210> 1
<211> 125
<212> PRT
<213> 人（Homo sapiens）
<400> 1
Gly Gln Phe Arg Val Ile Gly Pro Arg His Pro Ile Arg Ala Leu
1 5 10 15
Val Gly Asp Glu Val Glu Leu Pro Cys Arg Ile Ser Pro Gly Lys
20 25 30
Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Pro Pro Phe Ser
35 40 45
Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Gly Asp
50 55 60
Gln Ala Pro Glu Tyr Arg Gly Arg Thr Glu Leu Leu Lys Asp Ala
65 70 75
Ile Gly Glu Gly Lys Val Thr Leu Arg Ile Arg Asn Val Arg Phe
80 85 90
Ser Asp Glu Gly Gly Phe Thr Cys Phe Phe Arg Asp His Ser Tyr
95 100 105
Gln Glu Glu Ala Ala Met Glu Leu Lys Val Glu Asp Pro Phe Tyr
110 115 120
Trp Val Ser Pro Gly
125
Claims (2)
1.rhMOG30-154蛋白在制备诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的药物中的应用，其特征在于，所述rhMOG30-154蛋白的氨基酸序列如SEQ ID NO.1所示。
2.一种诱导食蟹猴实验性自身免疫性脑脊髓炎动物模型的药物，其特征在于，其含有权利要求1所述的rhMOG30-154蛋白作为活性成分。
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