CN116410324A - 一种肠道微生物脆弱拟杆菌毒素中和纳米抗体及其应用 - Google Patents
一种肠道微生物脆弱拟杆菌毒素中和纳米抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种肠道微生物脆弱拟杆菌毒素中和纳米抗体及其应用,通过深入研究直结肠癌肿瘤患者和乳腺癌患者体内的产肠毒素脆弱拟杆菌和其分泌的毒素BFT,采用噬菌体展示技术对BFT的纳米抗体进行表达;通过生物淘筛技术筛选出与抗原具有较高结合力的纳米抗体;得到了具有中和BFT毒性的纳米抗体,也称为Nb2.43。本发明表达并优化获得亲和力高且稳定均一的BFT纳米抗体蛋白,开辟了靶向BFT的纳米抗体的治疗直结肠癌和乳腺癌的新领域,具有良好的研究价值和应用前景。
Description
技术领域
本发明属于生物制药技术领域,涉及一种可以中和BFT的活性的抗体,具体涉及一种肠道微生物脆弱拟杆菌毒素中和纳米抗体及其应用。
背景技术
脆弱拟杆菌(Bacteroides fragilis)在人类肠道菌群中普遍存在,占肠道细菌总数的0.1%-0.5%,是腹腔脓肿以及血液感染样本中分离出来的最常见的厌氧菌。脆弱拟杆菌可以将糖、淀粉和纤维发酵成挥发性脂肪酸,被宿主细胞吸收和利用,为宿主提供营养和能量。脆弱拟杆菌是一种条件性致病菌,当宿主肠道屏障受损时,脆弱拟杆菌可以到胃肠区域以外的身体部位,导致中枢神经系统、颈部、口腔、肺部、腹部等器官组织的脓肿和感染。根据是否产毒素,脆弱拟杆菌可分为产肠毒素脆弱拟杆菌(Enterotoxigenic Bacteroidesfragilis,ETBF)和不产肠毒素脆弱拟杆菌(Non-toxigenic Bacteroides fragilis,NTBF)。ETBF可快速分泌脆弱拟杆菌毒素(BFT),导致溃疡性结肠炎、毒素介导的急性腹泻和菌血症的发生。研究发现,ETBF和pks+大肠杆菌在结肠粘膜的持续共定植,可促进家族性腺瘤性息肉病(FAP)患者肿瘤的形成。在对150位接受结肠镜检查的病人进行追踪,发现约有80%初次镜检携带ETBF的病人在12-15年后患有癌前病变,ETBF的定植可能是早期大肠癌发生的潜在标志。同时,ETBF可以在乳腺和肠道定植,迅速诱导乳腺上皮增生,促进乳腺癌的发生和转移进程[5]。BFT是产肠毒素脆弱拟杆菌(ETBF)中唯一公认的毒力因子,因此BFT可作为预测结直肠炎癌转化及乳腺癌等疾病发生、发展的生物标志物,可以通过研究靶向作用BFT的治疗手段来开发癌症等相关疾病的新型疗法。
目前,抗生素依然是治疗ETBF的主要方法,其中碳青霉烯类和甲硝唑是治疗脆弱拟杆菌感染最有效的方法,调查中发现甲硝唑耐药脆弱拟杆菌的报告率为0.5%至7.8%。脆弱拟杆菌对抗生素具有高度的耐药性,并含有丰富的抗生素耐药性机制。在过去的十年中,脆弱拟杆菌多药耐药分离株有所增加,其抗菌素耐药性(AMR)正在上升,尤其是碳青霉烯类和甲硝唑这两种被普遍用于治疗脆弱拟杆菌感染的抗生素。因此,开发新的治疗和检测方法至关重要。
BFT是一种Zn2+依赖的金属蛋白酶,大小约为20kDa。如图1所示,ETBF首先合成分子量约为45kDa的BFT前体蛋白,前体蛋白共由397个氨基酸残基组成,包括含有18个氨基酸残基的信号肽,含有193个氨基酸残基的前肽区和186个氨基酸残基的催化活性区。BFT前肽区通过天冬氨酸转换机制抑制其催化结构域活性,BFT前体蛋白经半胱氨酸蛋白酶fragipain加工后将具有催化活性的结构域(active BFT,aBFT)分泌到培养上清液中。研究表明,aBFT可以诱导肠上皮细胞的上皮钙黏蛋白(Epithelial cadherin,E-cadherin)胞外端裂解,破坏细胞间连接,导致肿瘤细胞浸润与转移。E-cadherin是一种I型经典钙黏蛋白,属于跨膜糖蛋白,属于钙黏蛋白家族中经典成员,参与介导细胞信号的转导以及细胞之间的黏附。E-cadherin功能出现异常,会导致细胞间的黏附功能出现紊乱,细胞间紧密连接被破坏,导致组织结构完整性受损。aBFT作为一种锌依赖性金属蛋白酶,可以在1min内特异性裂解E-cadherin胞外端,将E-cadherin裂解成~80kDa的可溶性胞外段和~40kDa的C端片段。研究表明,aBFT通过裂解E-cadherin促进炎症性肠病的发生以及诱导正常乳腺上皮细胞和乳腺癌细胞发生形态和功能变化以获得高度迁移和侵袭性的表型。肠上皮细胞E-cadherin被aBFT裂解,导致细胞间黏附功能受损,细胞间连接被破坏,肠上皮紧密连接状态受损,肠道通透性增强,引起肠上皮细胞内促肿瘤、促炎信号通路激活,促进肠道疾病的发生和发展。Liam Chung等人研究表明,aBFT可以触发结肠免疫细胞产生IL-17,引起黏膜免疫反应,IL17直接作用于结肠上皮细胞,进一步促进炎症相关信号通路NF-κb和STAT-3活化,继而诱导CXC趋化因子释放,从而招募更多免疫细胞参与炎症反应,促进结肠肿瘤发生。E-cadherin的胞内结构域通常与α-和β-catenin结合[8],当E-cadherin被aBFT裂解时,会促进β-catenin进入细胞核,结合LEF/TCF转录因子家族,启动下游靶基因c-myc的转录。ETBF也可以在乳腺中定植,通过分泌啊BFT激活乳腺癌和乳腺癌上皮细胞中的Notch1和β-catenin信号通路从而诱导乳腺增生,促进乳腺癌细胞的生长和转移。
纳米抗体是一种单域重链抗体,只包含一个重链可变区(VHH)和两条重链CH2与CH3区。VHH保留了全部的抗原结合能力,是最小的保留完整抗原结合片段,只有15kD。纳米抗体具有分子量小、制备周期短、高稳定性、免疫原性弱、高抗原结合性、组织穿透性好等特点,被广泛应用于分子影像、肿瘤诊断、免疫治疗、递送药物等领域。
目前与肠道细菌毒素相关的中和纳米抗体并未报道,针对aBFT筛选具有中和BFT毒性的纳米抗体,阻断aBFT与宿主细胞的结合,建立准确、有效和特异性的BFT的治疗方法,对ETBF引起的疾病进行治疗具有重大的研究价值和现实意义。因此,开发具有临床应用潜力的BFT中和纳米抗体有极大的现实意义和应用价值。
发明内容
本发明的目的在于提供一种肠道微生物脆弱拟杆菌毒素中和纳米抗体及其应用。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种肠道微生物脆弱拟杆菌毒素中和纳米抗体,该中和纳米抗体的重链包括3个抗原互补决定区,分别为CDR1、CDR2和CDR3;
其中,CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ IDNO:3所示。
优选地,该中和纳米抗体的重链还包括4个框架区,分别为FR1、FR2、FR3和FR4;其中,FR1、FR2、FR3和FR4的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6和SEQID NO:7所示。
进一步优选地,该中和纳米抗体的氨基酸序列如SEQ ID NO:8所示。
本发明还公开了一种编码上述的氨基酸序列如SEQ ID NO:8所示的肠道微生物脆弱拟杆菌毒素中和纳米抗体的核酸,该核酸的核苷酸序列如SEQ ID NO:9所示。
本发明还公开了一种原核表达载体,含有上述的肠道微生物脆弱拟杆菌毒素中和纳米抗体的核酸。
本发明还公开了一种原核宿主细胞,含有上述的原核表达载体。
本发明还公开了上述的肠道微生物脆弱拟杆菌毒素中和纳米抗体、核酸、原核表达载体或原核宿主细胞在制备治疗肿瘤的制剂中应用。
优选地,所述肿瘤为结直肠癌。
优选地,所述肿瘤为乳腺癌。
与现有技术相比,本发明具有以下有益效果:
本发明通过深入研究抗脆弱拟杆菌毒素,采用噬菌体展示技术对BFT纳米抗体进行表达,通过生物淘筛技术筛选出与抗原具有较高结合力的纳米抗体,为抗脆弱拟杆菌毒素有催化活性的结构域(active B.fragilis toxin以下简称aBFT)的特异性纳米抗体,也称为Nb2.43。本发明表达并优化获得亲和力高且稳定均一的aBFT中和纳米抗体蛋白,经ITC鉴定抗体结合活性,结果显示纳米抗体Nb2.43具有最高的结合活性的亲和力(KD)为5.59nM,是首个可以中和aBFT的特异性抗体,可以用于鉴定病人aBFT的阻断,作为结直肠癌以及乳腺癌治疗手段之一,具有较大的科学意义和临床应用价值。本发明开辟了检验人肠道细菌产肠毒素脆弱拟杆菌和脆弱拟杆菌毒素的治疗新领域,具有良好的研究价值和应用前景。
附图说明
图1为BFT金属蛋白酶蛋白质结构示意图;
图2为BFT1-sFL抗原重组蛋白纯化图;
图3用ELISA方法评价免疫效果图;
图4为用PCR方法随机挑取20个菌落计算插入率检测结果图;
图5为SDS-PAGE考马斯亮蓝染色结果验证纳米抗体大小和完整性实验结果;
图6为aBFT分子筛纯化后胶图;
图7为aBFT生物活性验证结果图;其中,(a)为control;(b)为使用aBFT处理;比例尺,50μm;
图8为Nb2.43与aBFT亲和力检测。
图9为Nb2.43对三种亚型aBFT均具有中和作用。
图10为Nb2.43影响aBFT对NCM460及HT29细胞中E-cadherin的表达;其中,(a)为NCM460细胞中E-cadherin蛋白表达量;(b)为HT29细胞中E-cadherin蛋白表达量;GAPDH作为内参蛋白;
图11验证体内小鼠中和抗体的功效,取小鼠结肠组织进行HE染色结果图。
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:
本发明利用噬菌体展示技术,从羊驼免疫的单域重链抗体中筛选能与靶重组蛋白BFT成熟体蛋白(aBFT)特异性结合的纳米抗体克隆。通用BFT重组蛋白对羊驼进行免疫,分离血液中的白细胞,利用噬菌体展示技术,构建噬菌体展示文库,经过3次连续生物淘筛法获得与BFT蛋白结合的噬菌体,经测序后和生物比对后,用酶联免疫吸附测定(enzyme-linked immune sorbent assay,ELISA)法筛选出抗aBFT的高亲和力纳米抗体。
1.BFT原核表达系统的构建和蛋白表达
分别构建了bft1-sFL不含信号肽全长原核表达质粒。通过NcoI和EcoRI限制性内切酶位点构建在pET28a载体上,同时在蛋白的N端添加6*HIS标签。Fpn通过NcoI和EcoRI限制性内切酶位点构建在pET28a载体上,同时在蛋白的C端添加6*HIS标签。通过使用IPTG诱导BFT重组蛋白表达,当细菌培养液OD600在0.6左右时,加0.4M IPTG,18℃低温诱导过夜。BFT1-sFL具有较好的可溶性。BFT1-sFL重组蛋白的体积排阻色谱的结果,如图2所示。
2.以BFT1-sFL为抗原的纳米抗体文库的构建
1)羊驼免疫血清的抗体检测
向一只5岁的健康雌性羊驼每周皮下注射100μg纯化的BFT1-sFL蛋白和免疫佐剂共6次。在第一次注射前和最后一次注射后第7天从颈静脉采集外周血,分离获得血清,用ELISA方法比较抗体滴度。向96孔板包被100μg/mL的BFT1-sFL蛋白,以PBS作为阴性对照。经洗板和封闭后,加入梯度稀释的免疫前和免疫后血清。以偶联HRP的山羊抗Llama抗体作为二抗,加入ABTS试剂进行反应。用酶标仪在405nm处测量吸光度值。结果参见图3,从图3中可以看出免疫后羊驼血清中靶向BFT1-sFL的抗体水平显著高于免疫前,证明以BFT重组蛋白混合免疫佐剂皮下注射的方法能够成功诱导羊驼的体液免疫反应,对实验动物的免疫达到了预期目的,可以进行后续构建文库等工作。
2)噬菌体文库的构建
于最后一次免疫后7天,从羊驼颈静脉收集100mL外周血,用Sepmate管和Lymphoprep分离外周血单个核细胞。用Trizol试剂从PBMCs中提取总RNA,用Random引物和逆转录酶合成cDNA。
用下述引物进行巢式PCR扩增VHH基因:
CALL001(5‘-GTCCTGGCTGTTCTCTCTCTCCAAGG-3’)
CALL002(5’-GGTACTGCTGTTTGAACTGTCC-3’)
以1%琼脂糖凝胶电泳,用快速凝胶提取编码重链抗体的基因片段(700bp)。
然后用下述引物作为第二次PCR模板:
VHH-for(5’-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3’)
VHH-Back(5‘-GATGTGCA GCAGGA GTCT GGRGGAG-3’)
这对引物是为VHH的框架1和框架4区域设计的,包含PstI和Eco91I酶切位点。第二轮PCR产物经电泳回收,用PstI、XbaI和Eco91I限制性内切酶酶切噬菌体载体pMES4,用PstI和Eco91I酶切第二轮PCR产物。用T4 DNA连接酶连接经酶切的pMES4和PCR产物。将重组载体转染大肠杆菌TG1感受态细胞,并在含氨苄西林的LB琼脂平板上进行培养。
随机挑取20个菌落,用下述载体引物进行菌落PCR,计算插入率:
GIII(5‘-CCACAGACCCCTCATAG-3’)
MP57(5’-TTATGCTTCCGGCTCGTATG-3’)。
经转化的TG1由M13K07辅助噬菌体感染在噬菌体上展示VHH片段。感染的细菌在含氨苄西林和卡那霉素的培养基中培养过夜。培养基下离心后,将上清液与PEG6000/NaCl混合分离噬菌体,之后将噬菌体颗粒重悬在1mL冰冷PBS中。VHH库的规模达到1.93*107/mL。对20个随机挑选的菌落进行PCR筛选,结果表明,大多数克隆插入了VHH基因,计算插入率(95%),结果参见图4,图4为琼脂糖凝胶电泳检测菌落PCR产物大小共1-20个菌落,条带分子量大小为700bp认定为插入了VHH片段的克隆,分子量大小400bp左右的条带认为转入了空质粒。M:DL2000核酸Marker。1-20为随机挑取的20个菌落。
3.纳米抗体筛选
1)生物淘筛
在包被100μL BFT1-sFL蛋白的96孔板上进行三轮生物淘筛以富集特异性结合BFT1-sFL的噬菌体。封闭后洗涤5次。将噬菌体文库加入抗原孔和阴性孔,室温孵育2h后用洗涤10-15次,用蛋白酶洗脱。分别取10μL从抗原孔和阴性孔中洗脱的噬菌体,梯度稀释并感染对数期大肠杆菌TG1,在含有氨苄西林的LB琼脂平板上划线。通过比较抗原孔和阴性孔噬菌体的效价,评价包含BFT1-sFL特异性结合VHHs的噬菌体的富集情况,如下表1所示。其余的噬菌体用于感染TG1并过夜培养,取菌液加入M13K07辅助噬菌体,重复上一节沉淀过程,将噬菌体亚文库扩增至后用于下一轮生物淘筛。经过三轮淘选,富集率达到4×104,如下表1所示:第三轮后抗原孔的滴度高于阴性孔1000倍以上,表明噬菌体文库中特异性结合BFT1-sFL的部分已充分富集,满足筛选阳性克隆的条件。
表1噬菌体的富集程度
c.f.u:菌落形成单位。
2)细菌胞质提取物ELISA
在含有氨苄西林的LB琼脂平板上分别培养第二轮和第三轮噬菌体子文库感染的大肠杆菌TG1细胞。随机挑取第二轮亚文库94个菌落和第三轮亚文库94个菌落,在含氨苄西林的TB培养基中培养。以1M IPTG在28℃过夜诱导。通过TES溶液提取胞质蛋白,以鼠源抗HIS抗体作为一抗,偶联HRP的羊抗鼠抗体作为二抗,以TMB试剂显色。用抗BFT抗体和偶联HRP的羊抗兔抗体作为阳性对照,以酶标仪检测吸光度值。将OD450的值比阴性孔的值高出2倍的克隆判定为阳性。将阳性克隆提取质粒进行测序,并按CDR3区进行分类。如表2所示:
表2噬菌体文库的筛选
4.纳米生物的表达、纯化以及鉴定
1)表达纯化
将特异性纳米抗体序列插入pHEN6c质粒,转染大肠杆菌WK6细胞。以1L TB培养基中,1mM IPTG诱导并提取HIS标记的重组纳米抗体,然后用Ni-NTA柱和固定化金属亲和层析纯化,将纳米抗体由咪唑透析至PBS。纳米抗体表达量良好为10.8mg/L。
2)SDS-PAGE分析
将40μL纯化后的纳米抗体加入10μL 5×loading buffer,100℃5min水浴。上样量为5uL至4%-15%的SDS-PAGE凝胶并电泳,用考马斯亮蓝染液染色2h。结果参见图5,图5可清晰地表明只有一个15kDa大小的条带。具体地,可见Nb2.43纳米抗体条带均位于15kD附近,符合测序结果。
5.获得BFT成熟体蛋白(aBFT)
使用6mg Fpn(C端6*His tag)酶切30mg BFT-sFL(N端6*His tag)蛋白。将BFT-sFL与Fpn混匀,于室温反应30min。将蛋白反应混合液使用Ni-NTA亲和层析柱进行纯化,在上样的同时收集穿出液。上样结束后,使用Buffer C洗脱,同时收集穿出液,直到UV280吸收值为Buffer C的吸收值。将所有穿出液进行浓缩,使用Superdex 75PG(GE Heathcare)分子筛进行进一步纯化。每管收集1mL,用NanoDrop 2000测定收集样品蛋白浓度。将收集的蛋白样品进行12% SDS-PAGE电泳检测,考马斯亮蓝染色,检验蛋白分离纯化效果,得到分子量吻合的高纯度aBFT蛋白,结果参见图6,图中A1、A5、B1、B4、B5、C1、C4、D1、D3为分子筛收集不同离心管蛋白样品。蛋白大小约为20kDa,与预期分子量吻合。
6.aBFT生物活性验证
aBFT纯化后需对其生物活性进行验证,HT29细胞对BFT较为敏感,可发生明显的形态学变化。HT29细胞以1.5×106个/孔的密度接种至12孔板中,在细胞汇合度为70%左右进行处理。使用3μg/mL aBFT在37℃条件下处理细胞20min后,出现明显的形态学变化,细胞间距增加,细胞变圆,说明纯化后aBFT具有生物活性。(结果参见图7)
7.Nb2.43与aBFT亲和力检测
等温滴定量热法(ITC)可以通过测量结合过程中的热传递,就能够准确地确定结合常数(Kd)、反应化学量(N)、焓(ΔH)和熵(ΔS)。提供了有关分子相互作用的完整热力学信息,ITC不仅可测定结合亲和力,还能阐明潜在分子相互作用的机制。ITC用Microcal ITC200calorimeter完成,反应在20℃下进行,Nb2.43和aBFT蛋白样品均将缓冲液置换到buffer A(20mM Tris-Hcl pH8.0,150mM NaCl,5% Glycerol)中,蛋白质样品通过Nanodrop分光光度计进行定量,Nb2.43蛋白浓度在200μM左右,aBFT蛋白浓度在20μM左右。实验数据用Microcal ITC 200calorimeter自带的Origin软件处理。
如图8所示,Nb2.43和aBFT的ITC结果显示,其解离常数(Kd)为5.59μM,ΔH为16kcal/mol,ΔG为-7.17kcal/mol,-TΔS为-23.2kcal/mol,结合反应化学量N为1,表示1个Nb2.43结合一个aBFT分子。
Nb2.43可以有效阻断aBFT-1水解细胞中E-cadherin蛋白,aBFT三种亚型氨基酸序列具有高度同源性,接下来对Nb2.43中和aBFT-2和aBFT-3蛋白活性的能力进行检测。首先将Nb2.43分别与aBFT-1、aBFT-2和aBFT-3预孵育以形成复合物,之后加入E-cadherin胞外端重组蛋白,将反应后样品使用12.5%的SDS-PAGE胶检测并进行考马斯亮蓝染色。结果表明,与aBFT单独处理组相比,Nb2.43可以显著阻断三种亚型aBFT对E-cadherin胞外端重组蛋白的水解作用,Nb2.43对aBFT三种亚型均具有中和作用(图9)。
8.BFT中和抗体—细胞实验
BFT可以诱导肠上皮细胞的E-钙黏蛋白(E-cadherin)胞外端裂解,破坏细胞间连接,导致肿瘤细胞浸润与转移。E-cadherin的裂解也可导致肠道通透性增加,肠道菌群代谢产物易位。此外,E-cadherin的裂解还可以激活β-catenin信号通路,从而加重肠道炎症反应。
为研究Nb2.43在细胞水平上的中和作用,将纳米抗体(0.1mg/mL)与aBFT(3ug/mL)进行混匀,4℃孵育30min以形成复合物。将BFT与纳米抗体混合物在37℃条件下,处理细胞10min,提取细胞总蛋白,采用Western Blotting的方法对E-cadherin蛋白表达量进行检测。
对人正常结肠上皮细胞NCM460及人结肠癌细胞HT29分别进行处理,与对照组相比,aBFT可显著降低NCM460及HT29细胞中E-cadherin蛋白表达量。Nb2.43可与aBFT形成复合物,可以阻断aBFT对NCM460及HT29细胞中E-cadherin蛋白的裂解,上调E-cadherin表达量(结果如图10所示)。
9.BFT中和抗体—小鼠实验
7周龄大,SPF级雌性C57BL/6小鼠被分为4组,分别是ETBF,NTBF,ETBF+Nb2.43(BFT中和抗体),ETBF+Nb119(同行对照纳米抗体)。小鼠在脆弱拟杆菌感染前一周在饮用水中加入100mg/L的克林霉素进行处理。一周后,将ETBF及NTBF制成1×1010CFU/ml细菌悬液,每只小鼠灌胃100uL,连续灌胃两天。第一次灌胃24h后,开始进行纳米抗体Nb2.43干预,纳米抗体干预组的每只小鼠尾静脉注射4mg纳米抗体,其余组尾静脉注射等体积PBS。一周给药4次,共给药一周。取小鼠结肠组织进行HE染色,结果如图11所示,可以看出,NTBF组小鼠结肠黏膜上层结构完整,无明显炎细胞浸润。ETBF诱导的结肠炎组小鼠结肠组织出现明显的炎细胞浸润。与Nb2.43处理组相比,Nb119(同行对照)处理组小鼠结肠组织有明显的炎性细胞浸润。与ETBF处理组相比,Nb2.43处理组小鼠结肠组织无明显的炎性细胞浸润。
综上所述,本发明从实验动物羊驼的免疫开始,构建了大容量纳米抗体的噬菌体展示文库。以生物淘筛(bio-panning)方法经过3轮筛选后,大量表达及纯化、我们得到了高亲和力的BFT纳米抗体,经ITC鉴定抗体结合活性,结果显示纳米抗体Nb2.43具有最高的结合活性的亲和力(KD)为5.59nM,是首个可以中和aBFT的特异性抗体,可以用于鉴定病人aBFT的阻断,作为结直肠癌以及乳腺癌治疗手段之一,具有较大的科学意义和临床应用价值。
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。
Claims (10)
1.一种肠道微生物脆弱拟杆菌毒素中和纳米抗体,其特征在于,该中和纳米抗体的重链包括3个抗原互补决定区,分别为CDR1、CDR2和CDR3;
其中,CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示。
2.根据权利要求1所述的肠道微生物脆弱拟杆菌毒素中和纳米抗体,其特征在于,该中和纳米抗体的重链还包括4个框架区,分别为FR1、FR2、FR3和FR4;其中,FR1、FR2、FR3和FR4的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7所示。
3.根据权利要求1所述的肠道微生物脆弱拟杆菌毒素中和纳米抗体,其特征在于,该中和纳米抗体的氨基酸序列如SEQ ID NO:8所示。
4.一种编码权利要求3所述的肠道微生物脆弱拟杆菌毒素中和纳米抗体的核酸,其特征在于,该核酸的核苷酸序列如SEQ ID NO:9所示。
5.一种原核表达载体,其特征在于,含有权利要求4所述的编码肠道微生物脆弱拟杆菌毒素中和纳米抗体的核酸。
6.一种原核宿主细胞,其特征在于,含有权利要求5所述的原核表达载体。
7.一种突变体,其特征在于,以权利要求3所述的肠道微生物脆弱拟杆菌毒素中和纳米抗体为前体,经随机突变、点突变或双特异性抗体改造,获得的特异性或靶向性增强的突变体。
8.权利要求1~3中任意一项所述的肠道微生物脆弱拟杆菌毒素中和纳米抗体、权利要求4所述的核酸、权利要求5所述的原核表达载体、权利要求6所述的原核宿主细胞或权利要求7所述的突变体在制备治疗肿瘤的制剂中应用。
9.如权利要求8所述的应用,其特征在于,所述肿瘤为结直肠癌。
10.如权利要求8所述的应用,其特征在于,所述肿瘤为乳腺癌。
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