CN102241776B - Rankl-tnf样区融合蛋白及其制备方法和应用 - Google Patents
Rankl-tnf样区融合蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种RANKL-TNF样区融合蛋白,其为RANKL-TNF样区蛋白与具有泛宿主性的Th2细胞表位片段的融合蛋白。实验表明,RANKL-TNF样区融合蛋白免疫小鼠产生的抗体具有结合RANKL和TNF-α的功效,可以用于治疗类风湿关节炎等疾病,发挥拮抗骨破坏和抑制炎症作用。本发明RANKL-TNF样区融合蛋白可以作为治疗性疫苗使用,为类风湿关节炎等疾病提供一种新的治疗途径和思路。本发明通过体外表达RANKL-TNF样区融合蛋白,该方法表达量高,可控性强,生产成本相对较低,且产物具有良好的免疫原性,容易实现大规模生产。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种RANKL-TNF样区融合蛋白,本发明还涉及该融合蛋白的制备方法以及在类风湿关节炎治疗性疫苗中的用途。
技术背景
类风湿关节炎(rheumatoid arthritis,RA)是一种慢性全身性自身免疫病,以破坏性关节炎为特征,病变特点为侵蚀性滑膜炎,以及由此造成的关节软骨和骨质破坏,最终导致关节畸形而致残。由于RA的患病率高、致残率高、病因尚未完全阐明,特别是RA给社会经济、劳动力造成了严重损失,因此,对RA治疗的研究具有重要的医学和社会经济意义。而RA又是一种难治性疾病,虽然现有治疗手段多种多样,但都不甚理想。本发明针对RA发病中骨破坏及慢性炎症反应这两个相关联的RA关键性病理学问题,研制RANKL-TNF样区融合蛋白,为RA的治疗开辟了一种新的思路。
RANKL是肿瘤坏死因子超家族(tumor necrosis factor superfamily,TNFSF)的成员,基因位于人13号染色体13q14,全长316个氨基酸,分子量大小为35~45kD,分三个亚型:RANKL1和RANKL2为跨膜型三聚体(trimeric transmembrane form),RANKL3为可溶型单体(solublemonomer),两种类型均可与诱饵受体骨保护素(osteoprotegerin,OPG)结合而失去生物活性,人鼠同源性为77%。RANKL为I型跨膜蛋白,由胞内、胞外和跨膜段组成,核心结构为胞外羧基端的158个氨基酸,由数条β型折叠构成,为TNF家族同源性结构域。RANKL主要分布在免疫***、骨骼***和循环***。参与骨破坏的RANKL主要由成骨/骨髓基质细胞、活化的T细胞、关节滑膜细胞和单核巨噬细胞分泌,通过自分泌和旁分泌方式与受体RANK结合发挥作用。RANKL的表达受到多种细胞因子的调控,如骨吸收因子、糖皮质激素、1α,25(OH)2-VD3、IL-1、IL-6、IL-11、TNF-α、PGE2和PTH等,这些因子都可诱导成骨细胞表达RANKL,从而促进骨吸收。RANKL的发现使我们对破骨细胞(osteoclast,OC)分化、活化、增殖有了更进一步的认识。RANKL的受体RANK(receptor activatorof nuclear factor)是肿瘤坏死因子受体超家族(tumor necrosis factor receptorsuperfamily,TNFRSF)成员,基因定位于18q22.1,是616个氨基酸的肽段,属I型跨膜蛋白,有一个短的含有21个氨基酸的穿细胞膜基团和一个383个氨基酸残基的细胞内羧基末端,人鼠同源性为60%。主要表达在单核巨噬细胞***,包括破骨细胞及前体细胞、淋巴细胞、内皮细胞、成纤维细胞。RANKL在RA患者关节腔表达量增加并与破骨细胞及其前体细胞表面的RANK结合,RANK胞内域构象改变与其衔接蛋白肿瘤坏死因子受体相关因子6(TNF receptor-associated factor 6,TRAF6)结合后使其活化;活化的TRAF6作为一个多功能的第二信使(second messager)使转录因子NF-κB或***原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)激活,其中MAPK又包含了应激活化蛋白激酶(c-Jun N-terminalkinases/stress-activated protein kinase,JNK)和胞外信号调节激酶(extracellular signal-regulated kinases,ERK)两条途径。JNK能进一步激活c-Jun和活化蛋白1(AP-1),影响破骨细胞和T细胞核内因子的活性。ERK是c-Fos表达与否的调控因子,后者是破骨细胞分化中一个重要的转录因子。NF-κB作为一个二聚体转录因子,激活后由胞浆进入胞核识别DNA序列的κB位点;钙信号启动核因子活化的T细胞c1(nuclear factor ofactivated T cell c1,NFATc1)并与启动子区结合,使破骨细胞分化相关基因转录表达,破骨前体细胞增殖、分化、成熟为有功能的破骨细胞,使骨吸收增加。同时,破骨细胞表面也存在RANK,与RANKL结合后使破骨功能增强。与此同时,表达在活化的T细胞上的RANKL与自身或DC上RANK结合,能激活免疫***。体外实验证明,T细胞产生的RANKL能直接启动破骨细胞的增殖;ctla4-/-基因敲除鼠能抑制活化T细胞对骨密度的破坏。免疫***与骨***之间的恶性循环是RA骨破坏持续存在的原因。
RA是慢性进展的***性炎症,许多炎性因子参与了类风湿的病理过程,TNF-α是RA发病机理中居中心地位的炎症细胞因子之一,它参与了RA的发生发展过程。TNF-α可诱导内皮细胞表达黏附分子(inter-cellularadhesion molecule,ICAM),促进白细胞与血管内皮黏附渗透导致局部的炎症。此外,TNF-α刺激滑膜纤维母细胞和软骨细胞产生***素E2(prostaglandin E2,PGE2)和胶原酶,促进骨质破坏和骨的吸收及纤维母细胞的增生,抑制骨胶原的合成。TNF-α也能促进人软骨细胞分泌纤维蛋白溶酶激活剂,使纤维蛋白溶酶原变成纤维蛋白溶酶而加快关节炎的损失过程,TNF-α增加了滑膜内皮细胞及成纤维细胞生长因子的释放而促进血管翳的形成。TNF-α还可促使滑膜细胞、巨噬细胞、纤维母细胞和软骨细胞产生IL-1、IL-8,加之TNF-α本身而加重组织损伤。Saxne等学者报告50%的RA患者血清中TNF-α水平升高,且TNF-α升高的多为关节明显肿胀、压痛及血沉快的活动期患者。提示RA关节炎症越重,TNF-α水平越高。TNF-α在活动性RA中参与三种主要病理过程:①激活血管内皮细胞,增强内皮细胞ICAM的表达,在关节炎时血液中白细胞正是通过与ICAM的相互作用被汇集到关节腔的。②刺激***细胞和多形核细胞产生PGE等小分子炎症介质。③通过刺激滑膜细胞和软骨细胞,使破骨细胞减少糖蛋白合成,增加糖蛋白降解,并产生胶原酶和其他中性蛋白酶、释放骨钙等,从而导致骨和软骨的破坏。可溶性TNFR可有效地减轻佐剂性关节炎的病理改变。
研究发现,以RANKL为阻断目标的药物可以减轻RA骨和软骨的破坏,用RANKL单克隆抗体或RANK-Fc能显著改善肿瘤转移性溶骨和绝经后骨质疏松的症状。在一项阿根廷的随机双盲试验中,应用人源化的单克隆抗体AMG-162治疗绝经后骨质疏松病人的研究表明,RANKL单抗能显著增加骨量,减轻骨痛等症状,未见明显不良反应,但缺点是对炎症性滑膜炎无效,必须与抗TNF-α的其他制剂联合应用。TNF-α拮抗剂是临床上抑制RA炎症反应疗效肯定的制剂,但有1/3的患者对目前上市的三种拮抗剂无效,而且由于TNF-α拮抗剂的价格比较昂贵,很难得到普遍应用。
发明内容
本发明的第一个目的在于针对上述不足提供一种RANKL-TNF样区融合蛋白。
本发明的第二个目的在于提供上述RANKL-TNF样区融合蛋白的制备方法。
本发明的第三个目的在于提供上述RANKL-TNF样区融合蛋白在治疗类风湿关节炎的生物制剂/疫苗制备中的用途。
本发明RANKL-TNF样区融合蛋白,其为RANKL-TNF样区蛋白与具有泛宿主性的Th2细胞表位片段的融合蛋白。
RANKL和TNF-α是RA骨损伤和炎症持续存在的关键因子,同时,RANKL为TNFSF家族成员,RANKL的TNF样区与TNF-α的氨基酸序列有高度的相似性,本发明将RANKL中与TNF-α相似性高的两段区域作为多肽疫苗的候选表位,但RANKL为自体成分,因此天然状态下很难产生抗体。本发明通过将RANKL与TNF-α的非相似区域用泛宿主性的Th2细胞表位取代,从而增强了其免疫原性。这种泛宿主性的Th2细胞表位可以是HIV-I的BRU株逆转录酶RT(414-424aa)的基因序列GAATGGGAGTTCGTAAACACCCCACCTCTCGTC(氨基酸序列EWEFVNTPPLV),或者是其他的泛宿主性Th2细胞表位,例如:OVA、BSA、PPD等。
术语“泛宿主性”是指广泛的宿主适应性。
在本发明实施例中,所述RANKL-TNF样区融合蛋白的氨基酸序列如SEQ ID No.2或4;本领域技术人员可根据该片段,在不影响其活性的前提下,取代、缺失和/或添加一个或几个氨基酸,得到所述蛋白的突变序列。例如,将第6位的丝氨酸替换为酪氨酸,或者将第77位的赖氨酸缺失,或者在第118位添加芳香族氨基酸。因此,RANKL-TNF样区融合蛋白还包括SEQ ID No.2或4所示氨基酸序列经取代、替换和/或增加一个或几个氨基酸,具有同等活性的由SEQ ID No.2或4衍生得到的蛋白质。
本发明还包括编码上述融合蛋白的基因。在本发明实施例中,编码该融合蛋白基因的核苷酸序列如SEQ ID No.1或3所示。应理解,考虑到密码子的简并性以及不同物种密码子的偏爱性,本领域技术人员可以根据需要使用适合特定物种表达的密码子。
可将本发明上述基因与表达载体可操作地连接,得到能够表达本发明融合蛋白的重组表达载体,进而可以将该表达载体导入宿主细胞,得到表达RANKL-TNF样区融合蛋白的基因工程菌。本发明所使用的表达载体可以选用各种已知的原核表达载体。
本发明还提供一种制备上述融合蛋白的方法,包括如下步骤:将编码RANKL-TNF样区融合蛋白的基因克隆到表达载体中,将所述表达载体转化宿主细胞,筛选阳性克隆,经菌体培养和诱导表达后,分离纯化得到该融合蛋白。
在本发明实施例中,通过构建重组基因,与pET-28a表达载体连接,转化大肠杆菌BL21(DE3),表达融合蛋白,并通过分子筛或离子交换层析等方法对融合蛋白进行纯化,采用SDS-PAGE、Western-blot等对融合蛋白进行理化性质鉴定。
在本发明的一个优选的实施例中,在合适的条件下发酵培养工程菌的步骤包括:
将测序正确的重组克隆质粒转化大肠杆菌BL21(DE3)感受态细胞,挑选单菌落接种于10mL LB培养基(含30mg/L的卡那霉素),37℃,250rpm振荡培养过夜。次日取30mL过夜培养物转接于1L LB培养基(含30mg/L的卡那霉素)中,37℃,250rpm培养至OD600至0.5,加入IPTG使之终浓度为0.4mM,37℃,250rpm继续振荡培养4.5h后离心收集菌体。
取培养后收集的大肠杆菌菌体,用预冷的PBS洗涤菌体6次,超声重悬10min(超4s停6s,功率为40%)。每克湿菌体加10倍体积的细菌细胞蛋白裂解液重悬,加入溶菌酶至1mg/mL,同时加入蛋白酶抑制剂PMSF至1mM,冰上放置30min后,在冰浴中超声裂菌10min,12,000rpm离心10min收集沉淀。再分别用TE缓冲液、1%trixton-100各洗涤一次,将沉淀溶于PBS中超声重悬菌体(5mL/g湿菌体),同时缓慢滴加等体积的8M尿素溶液,磁力搅拌子搅拌30min,离心后沉淀用4M尿素溶液洗涤1次,沉淀重悬于含4M尿素的溶解缓冲液中,离心取上清经0.45μm的微孔滤膜过滤获得含目的融合蛋白的变性液。亲和层析法(Ni-NTA·His预装柱5mL)纯化融合蛋白。根据BCA蛋白定量法将纯化的蛋白变性液浓度稀释到0.5mg/mL。按尿素浓度逐步递减(4M-3M-2.5M-2M-1.5M-1.2M-0.8M-0.5M-0.2M-0M)的方法配好复性液(4M尿素,50mM Tris,0.5mM EDTA,50mM NaCl,3mM还原型谷胱甘肽,1mM氧化型谷胱甘肽,10%(V/V)甘油,1%甘氨酸,PBS,pH7.6),将稀释的蛋白变性液置于透析袋内,按复性内、外液比例不大于1∶20的比例4℃缓慢搅拌透析复性。根据透析过程中蛋白是否析出以及析出量的多少确定最佳复性透析条件,约4个小时更换一次尿素浓度逐渐降低的透析液,最后在pH7.6的PBS溶液中透析过夜,其间更换一次PBS溶液,对蛋白变性液进行透析复性。
实验表明,RANKL-TNF样区融合蛋白免疫小鼠产生的抗体具有结合RANKL和TNF-α的功效,可以用于治疗类风湿关节炎等疾病,发挥减轻骨破坏和抑制炎症作用,从而可以用于制备类风湿关节炎的疫苗。进而,本发明还包括一种治疗类风湿关节炎的治疗性疫苗,其为有效剂量的上述RANKL-TNF样区融合蛋白与生物制剂/疫苗学上可接受的载体制成。
本发明RANKL-TNF样区融合蛋白为类风湿关节炎等疾病提供了一种新的治疗途径和思路。本发明通过体外表达RANKL-TNF样区融合蛋白,表达量高,可控性强,生产成本相对较低,且表达产物具有良好的免疫原性,容易实现大规模生产。
附图说明
图1显示本发明所选用的表达载体及重组后表达载体的结构示意图;其中,图1(a)为pET-28a(+)质粒示意图;图1(b)为pET-28a(+)-RTFP-1重组质粒示意图;图1(c)为pET-28a(+)-RTFP-2重组质粒示意图;图1(d)为pET-28a(+)-RTFP-3重组质粒示意图。
图2为RANKL-TNF样区融合蛋白PCR产物琼脂糖凝胶电泳图谱;其中,图2(a)为融合蛋白RTFP-1重组表达质粒的构建结果,泳道M1,M2:分子量Marker;泳道1:RANKL的全基因克隆质粒(BC117286,1097bp;pDONR223,5004bp);泳道2,3:目的基因的AB,CD片段;泳道4:目的基因全长(443bp);泳道5:目的基因全长的双酶切片段(Sal I、Nde I);泳道6:pET-28a(+)质粒;泳道7:pET-28a(+)质粒双酶切(Sal I、Nde I);泳道8:含目的基因的重组表达质粒(pET-28a(+)-RTFP-1);泳道9,10:重组表达质粒的PCR鉴定(pET-28a(+),pET-28a(+)-RTFP-1);泳道11:pET-28a(+)-RTFP-1重组表达质粒的双酶切鉴定(Sal I、Nde I);
其中,图2(b)为融合蛋白RTFP-2重组表达质粒的构建结果,泳道M1,M2:分子量Marker;泳道1:RANKL的全基因克隆质粒(BC117286,1097bp;pDONR223,5004bp);泳道2,3,4:目的基因的AB、CD、CE片段;泳道5:目的基因全长(443bp);泳道6:目的基因全长的双酶切片段(Sal I、Nde I);泳道7::pET-28a(+)质粒;泳道8:pET-28a(+)质粒双酶切(Sal I、Nde I);泳道9:含目的基因的重组表达质粒(pET-28a(+)-RTFP-2);泳道10,11:重组表达质粒的PCR鉴定(pET-28a(+),pET-28a(+)-RTFP-2);泳道12:pET-28a(+)-RTFP-2重组表达质粒的双酶切鉴定(Sal I、Nde I);
其中,图2(c)融合蛋白RTFP-3重组表达质粒的构建结果,泳道M1,M2:分子量Marker;泳道1,2:目的基因的全长(410bp);泳道3:pET-28a(+)质粒;泳道4,5:含目的基因的重组表达质粒(pET-28a(+)-RTFP-3)。
图3为RANKL-TNF样区融合蛋白SDS-PAGE电泳图谱;其中,泳道M1:分子量Marker;泳道1,3,5,7:未加IPTG诱导的重组表达质粒(pET-28a(+),pET-28a(+)-RTFP-1,pET-28a(+)-RTFP-2,pET-28a(+)-RTFP-3;37℃,250rpm);泳道2,4,6,8:加入IPTG诱导的重组表达质粒(pET-28a(+),pET-28a(+)-RTFP-1(17.9KDa),pET-28a(+)-RTFP-2(19.9KDa),pET-28a(+)-RTFP-3(16.6KDa);37℃,250rpm,IPTG 0.4mM)。
图4显示的是Western-blot分析RANKL-TNF样区融合蛋白;
其中,图a为用抗人RANKL多克隆抗体检测融合蛋白,泳道1:RTFP-1融合蛋白;泳道2:RTFP-2融合蛋白;泳道3:RTFP-3融合蛋白;
其中,图b为用抗人TNF-α多克隆抗体检测融合蛋白,泳道1:阳性对照(rhTNF-α,17.5KDa);泳道2:阴性对照(pET-28a(+)质粒);泳道3:RTFP-1融合蛋白;泳道4:RTFP-2融合蛋白;泳道5:RTFP-3融合蛋白。
图5纯化复性后的RANKL-TNF样区融合蛋白SDS-PAGE电泳图谱;其中,泳道M1:分子量Marker;泳道1:RTFP-1融合蛋白;泳道2:RTFP-2融合蛋白;泳道3:RTFP-3融合蛋白。
图6显示的是RANKL-TNF样区融合蛋白的安全性鉴定;
其中,图6(a)是破骨细胞生成试验,其中A.阳性对照组:(M-CSF10ng/mL+RANKL 40ng/mL);B.融合蛋白RTFP-1实验组:(M-CSF10ng/mL+融合蛋白RTFP-1 30μg/mL);C.融合蛋白RTFP-2实验组:(M-CSF 10ng/mL+融合蛋白RTFP-2 30μg/mL);
其中,图6(b)是L929细胞凋亡试验,其中A.空白对照组:(L929);B.阳性对照组:(L929+TNF-α20ng/mL);C.融合蛋白RTFP-1实验组:(L929+融合蛋白RTFP-1 30ug/mL);D.融合蛋白RTFP-2实验组:(L929+融合蛋白RTFP-2 30ug/mL);
其中,图6(c)是融合蛋白对L929细胞凋亡活性的测定结果。
图7显示的是RANKL-TNF样区融合蛋白刺激机体产生抗体的滴度及持续时间,其中图7(a)是用rhRANKL作为包被抗原检测免疫小鼠血清中产生的中和抗体滴度及持续时间;图7(b)是用rhTNF-α作为包被抗原检测免疫小鼠血清中产生的中和抗体滴度及持续时间。
图8显示的是RANKL-TNF样区融合蛋白刺激机体产生抗体中和rhTNF-α和rhRANKL的能力,其中图8(a)是rhTNF-α所致的恶液质小鼠的各组生存率检测;图8(b)是用rhRANKL所致骨钙释放后各组小鼠的血钙水平测定。
图9显示的是RANKL-TNF样区融合蛋白刺激机体产生抗体对胶原诱导关节炎(Collagen-Induced Arthritis,CIA)小鼠的保护作用评估,其中图9(a)是各组小鼠的发病率;图9(b)是各组小鼠的关节肿胀度测定;图9(c)是各组小鼠的关节评分;图9(d)是各组小鼠Micro-CT的骨侵蚀程度变化。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 RANKL-TNF样区融合蛋白原核表达载体的构建
依据RANKL-TNF样区重组片段(RANKL P171-234、P246-309,HIV-1的BRU株逆转录酶RT(414-424aa))基因序列和pET-28a(+)表达载体的特征设计引物(见下表),以RANKL全长cDNA为模板(Gen Bank NO:BC117286,1097bp;pDONR223,5004bp),保留了pET-28a自带的His标签,采用重组PCR的方法连接三者的基因片段,其中将HIV-I的BRU株逆转录酶RT(414-424aa)的基因序列插在两个RANKL-TNF样区基因片段的中间,构建得到表达载体为pET-28a(+)-RTFP-1;将HIV-I的BRU株逆转录酶RT(414-424aa)的基因序列插在两个RANKL-TNF样区基因片段的末端,构建得到表达载体为pET-28a(+)-RTFP-2,并将上述两个RANKL-TNF样区基因片段直接拼接的野生型突变体作为对照得到的表达载体为pET-28a(+)-RTFP-3。相应的融合基因序列如SEQ ID No.1或3、5所示。PCR反应条件:98℃预变性30s,按下述参数循环30次:96℃变性5s,55-65℃退火15s,72℃延伸15s,最后72℃延伸10min。PCR产物在1%的琼脂糖凝胶中进行电泳观察其大小,通过重组表达质粒的PCR鉴定和双酶切鉴定,见图2,PCR扩增出预期大小的目的条带,且双酶切鉴定重组质粒含有的目的基因片段为预期大小,说明本发明成功构建了RANKL-TNF样区融合蛋白的原核表达载体。
表1 相关引物
实施例2 RANKL-TNF样区融合蛋白在大肠杆菌中的诱导表达
将测序正确的重组克隆质粒转化大肠杆菌BL21(DE3)感受态细胞,挑选单菌落接种于10mL LB培养基(含30mg/L的卡那霉素),37℃,250rpm振荡培养过夜。次日取30mL过夜培养物转接于1L LB培养基(含30mg/L的卡那霉素)中,37℃,250rpm培养至OD600至0.5,加入IPTG使之终浓度为0.1mM,37℃,250rpm继续振荡培养4.5h后离心收集菌体,SDS-PAGE及Western-blot分析表明经0.4mMIPTG诱导后可得到大量的融合蛋白,见图3,4。
实施例3 RANKL-TNF样区融合蛋白的纯化与复性
阳性克隆菌株于0.4mM IPTG,37℃诱导培养4.5h后,4℃,6000rpm离心15min,收集菌体,用预冷的PBS洗涤菌体6次,超声重悬10min(超4s停6s,功率为40%)。每克湿菌体加10倍体积的细菌细胞蛋白裂解液重悬,加入溶菌酶至1mg/mL,同时加入蛋白酶抑制剂PMSF至1mM,冰上放置30min后,在冰浴中超声裂菌10min,12,000rpm离心10min收集沉淀。再分别用TE缓冲液、1%trixton-100各洗涤一次,将沉淀溶于PBS中超声重悬菌体(5mL/g湿菌体),同时缓慢滴加等体积的8M尿素溶液,磁力搅拌子搅拌30min,离心后沉淀用4M尿素溶液洗1次,沉淀重悬于含4M的溶解缓冲液中,离心取上清经0.45μm的微孔滤膜过滤获得含目的融合蛋白的变性液。亲和层析法(Ni-NTA·His预装柱5mL)纯化目的蛋白。根据BCA蛋白定量法将纯化的蛋白变性液浓度稀释到0.5mg/mL。按尿素浓度逐步递减(4M-3M-2.5M-2M-1.5M-1.2M-0.8M-0.5M-0.2M-0M)的方法配好复性液(4M尿素,50mM Tris,0.5mM EDTA,50mM NaCl,3mM还原型谷胱甘肽,1mM氧化型谷胱甘肽,10%(V/V)甘油,1%甘氨酸,PBS,pH7.6),将稀释的蛋白变性液置于透析袋内,按复性内、外液比例不大于1∶20的比例4℃缓慢搅拌透析复性。根据透析过程中蛋白是否析出以及析出量的多少确定最佳复性透析条件,约4个小时更换一次尿素浓度逐渐降低的透析液,最后在pH7.6的PBS溶液中透析过夜,其间更换一次PBS溶液,对蛋白变性液进行透析复性,复性后的RANKL-TNF样区融合蛋白SDS-PAGE电泳图见图5,说明采用上述方法该融合蛋白可成功复性。
实施例4安全性实验:RANKL-TNF样区融合蛋白的安全性鉴定
建立以M-CSF和RANKL为诱导分化因子的骨髓细胞分化为破骨细胞的诱导培养体系。取6-15W的BABL/c雌性SPF级小鼠,于无菌条件下用注射器冲洗小鼠四肢骨的骨髓腔获得骨髓细胞,70nm细胞筛过滤后用RPMI1640培养基和10%FBS重悬骨髓细胞制成单细胞悬液(玻片计数细胞密度为2.5×106/mL),于25cm2培养瓶中在37℃、5%CO2饱和湿度条件下原代培养小鼠骨髓细胞,加入M-CSF至终浓度为25ng/mL后培养24h,次日收集细胞悬液,即为M-CSF依赖性非附着性骨髓单核细胞。1000rpm,常温离心5min,弃上清,PBS洗两遍。用RPMI1640培养基和10%FBS重悬细胞,计数并调整细胞浓度为2.5×106/mL,将细胞分别接种于6孔板和96孔板中。通过促进骨髓单核细胞分化为破骨细胞的破骨细胞生成试验,将实验分为RANKL-TNF样区重组多肽组(含25ng/mL的M-CSF+30μg/mL的融合蛋白RTFP-1/2)和天然人RANKL阳性对照组(含25ng/mL的M-CSF+40ng/mL RANKL),每组12个样本,TRAP染色观察融合蛋白能否刺激骨髓单核细胞分化为破骨细胞,结果显示,融合蛋白无天然RANKL的促进破骨细胞生成的生物学活性,见图6(a)。
建立L929细胞凋亡培养体系,L929细胞系为首都医科大学细胞生物学教研室馈赠,常规复苏细胞,用RPMI1640培养基和10%FBS重悬细胞,并计数调整细胞浓度为1×106/mL,将细胞分别接种于6孔板和96孔板中,在37℃、5%CO2饱和湿度条件下贴壁培养24h,将实验分为RANKL-TNF样区重组多肽组(含30μg/mL的融合蛋白)、天然人TNF-α细胞因子组(含20ng/mL的rhTNF-α),及实验对照组(只用上述完全培养基培养),分别加入上述融合蛋白及细胞因子继续培养12h,6孔板每组3个复孔,光镜下观察融合蛋白能否促进L929细胞凋亡的形态变化;96孔板每组12个样本,用结晶紫染色法于570nm波长下测定细胞凋亡率,结果显示,与天然人TNF-α细胞因子组相比,融合蛋白无促进L929细胞凋亡的活性,见图6(b)。
上述实验证明,融合蛋白虽为RANKL-TNF样区的变体,但无天然RANKL的骨破坏作用及天然TNF-α的促凋亡作用,安全性能良好,可进一步用于体内试验。
实施例6动物免疫实验
融合蛋白免疫6~8w雌性BABL/c小鼠(SPF级),按体重将小鼠分为实验对照组和融合蛋白RTFP-1、RTFP-2、RTFP-3组,腹部皮下多点免疫小鼠(初次免疫后记为第0d,分别于21d、42d、152d加强免疫),每只小鼠每次免疫剂量为200ug(100μl),初次免疫用与免疫原等量的完全弗氏佐剂(CFA)乳化,加强免疫用等量不完全弗氏佐剂(IFA)乳化。不同时间点(0d,32d,49d,56d,63d,70d,84d,112d,142d,163d,170d,177d)眼眶取血后用间接ELISA法检测小鼠体内产生中和抗体的能力,分别用rhTNF-α(200ng/mL)和rhRANKL(200ng/mL)包被ELISA板,4℃过夜,并用1%BSA37℃封闭1h,待检血清1∶1000稀释,HRP标记的抗体1∶20,000稀释。血清抗体滴度测定结果显示,含Th2细胞表位的融合蛋白组抗体产生水平明显高于实验对照组,且明显高于无Th2细胞表位的融合蛋白对照组(数据如图7所示)。
实施例7抗体体内中和实验
参照实施例6所述方法,在42d加强免疫后1W,RTFP-2融合蛋白组和实验对照组小鼠尾静脉给予rhTNF-α(20ug/只),隔天注射,连续三次,复制小鼠恶液质模型。首次注射后每天监测各组小鼠的生存率,结果显示,含Th2细胞表位的RTFP-2融合蛋白组小鼠的存活率明显高于实验对照组[数据如图8(a)所示];RTFP-2融合蛋白和实验对照组小鼠尾静脉给予rhRANKL(10ug/只),连续注射3d,于注射前及每次注射后3h及首次注射后7d眼眶采血,测定血清钙离子水平,结果显示,含Th2细胞表位的RTFP-2融合蛋白组小鼠可拮抗rhRANKL所致的血钙水平升高[数据如图8(b)所示]。
实施例8疫苗对胶原诱导关节炎小鼠的保护作用实验
融合蛋白免疫5W雄性DBA-1小鼠(SPF级),方法及免疫流程参照实施例6。末次免疫后1W,除实验对照组小鼠外,各组小鼠距尾根部1.5cm处皮内免疫牛二型胶原(CII)与等量含4mg/mLBCG的CFA的乳化物(200ugCII,100ul/只),3W后相同方法加强免疫(100ugCII+IFA,100ul/只),复制CIA小鼠模型。加强免疫后隔天监测各组小鼠的发病率,并进行关节评分[评分标准:0=无关节炎症状;1=足爪或一个脚趾肿和(或)红;2=累及两个关节的肿和(或)红;3=两个以上的关节肿和(或)红;4=严重的关节炎症状累及整个足爪或脚趾],同时用0.02mm量程的游标卡尺测定四肢关节的肿胀度变化至末次免疫后50d;末次免疫CII后50d断颈处死小鼠,Micro-CT断层扫描,评估各组小鼠的骨侵蚀程度。结果显示,含Th2细胞表位的RTFP-2融合蛋白组小鼠发病率降低,且疾病症状轻,骨损伤不明显(p<0.05,数据如图9所示)。
Claims (8)
1.RANKL-TNF样区融合蛋白,其为RANKL-TNF样区蛋白与具有泛宿主性的Th2细胞表位片段的融合蛋白,所述融合蛋白为由SEQ ID No.4所示氨基酸序列组成的蛋白。
2.编码权利要求1所述融合蛋白的基因。
3.如权利要求2所述的基因,其核苷酸序列如SEQ ID No.3所示。
4.含有权利要求2或3所述融合蛋白编码基因的表达载体。
5.含有权利要求4所述融合蛋白表达载体的宿主细胞。
6.一种制备权利要求1所述融合蛋白的方法,其包括如下步骤:将权利要求2或3所述的基因克隆到表达载体,将所述表达载体转化宿主细胞,筛选阳性克隆,经菌体培养和诱导表达后,分离纯化得到该融合蛋白。
7.权利要求1所述的融合蛋白在制备治疗类风湿关节炎的疫苗中的应用。
8.一种治疗类风湿关节炎的疫苗,其为有效剂量的权利要求1所述的融合蛋白与生物制剂/疫苗学上可接受的载体制成。
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