CN111748571A - 一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法 - Google Patents
一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法 Download PDFInfo
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Abstract
本发明公开了一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,通过基因工程的方法,对枯草芽孢杆菌进行改造,得到可用于生产纳米抗体的多功能稳定的平台,同时也可进一步将工程化后的枯草芽孢杆菌制为芽孢保存备用。相较于大肠杆菌法和LCE法,本发明中生产纳米抗体的方法,具有所得产品的毒素水平低、纯化工艺更简单、生产规模易于扩大、可长期保存以及在极端环境下进行生产等优点。
Description
技术领域
本发明涉及生物医药领域,具体是一种利用枯草芽孢杆菌生产纳米抗体的方法。
背景技术
纳米抗体是1993年由比利时科学家在羊驼体内发现的,存在于骆驼外周血液中的一种特殊抗体,因仅由一个最小抗原结合片段的结构域组成,含有天然重链可变区域,分子的长度在纳米级别而得名。
这种源自骆驼科动物的单链可变抗体片段的纳米抗体,与单克隆抗体相比,小分子纳米抗体的制备相对容易和简单。此外,由于纳米抗体易于进行基因操作,从而可以形成单价、双价、双特异及多价抗体,形成融合蛋白进行靶向治疗,同时由于纳米抗体具有分子量小,可溶性高,亲和力好,相对稳定,穿透力较强等优势,纳米抗体在全身成像和开发用于治疗多种疾病(包括但不限于炎症,癌症,骨骼疾病,血液学和传染性疾病)的新药物中已证明具有翻译潜力,在临床治疗与诊断,医药研发及食品科学等领域均具有较广阔的前景及应用。
生产包括纳米抗体在内的各类生物制剂的主力为细菌,酵母,植物和哺乳动物细胞之类的活细胞系统,但是,大肠杆菌的细胞内表达来生产纳米抗体这种方法需要漫长的步骤来释放细胞内蛋白质以进行纯化以及去除内毒素,大大延长下游的加工时间。近几年,从活细胞制备的冻干细胞提取物(LCE)使药物生产分散化和室温储存成为可能,可作为上述方法的备选方案,但是细胞提取物不能复制,因此难以扩大规模;一些LCE系统使用大肠埃希氏菌,因此必须采取严格的程序去除内毒素污染;细胞裂解物中蛋白质和小分子的混合物需要大量的下游产物纯化;在无细胞蛋白表达系统中存在批次间的变异性。
枯草芽孢杆菌的内毒素水平极低,可显著简化下游纯化步骤,但枯草芽孢杆菌在表达外源蛋白的同时,自身也会向胞外分泌大量的蛋白酶,使外源蛋白无法稳定存在。很多真核蛋白在枯草芽孢杆菌中的Sec依赖性分泌途径产生的质量较差,虽然已经开发出相应的菌株来减轻该问题,但是由于目标蛋白的错误折叠使得分泌的某些真核蛋白仍存在质量问题。
发明内容
本发明使用枯草芽孢杆菌作为微生物工厂生产纳米抗体,属于大肠杆菌法和LCE法生产纳米抗体的可替代方案,在解决了现有种种问题,如目标蛋白错误折叠而导致产品蛋白的质量及产量下降等,该方法更具优势:枯草芽孢杆菌所含的毒素水平低,下游的纯化工艺更简单,且生产规模易于扩大;将含有纳米抗体融合基因的枯草芽孢杆菌转化为芽孢,可以稳定地长期保存,且可在极端环境下进行生产等。
本发明具体采用的技术方案为:
将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的步骤如下:
A1:构建融合基因:所述融合基因包括纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和用于在含三乙酸(NTA)的层析柱上进行金属亲和层析的6×组氨酸标签(His标签);合成DNA片段,并对编码所述DNA片段的密码子进行优化;
A2:扩增所述DNA片段,并纯化,得到扩增产物;
A3:将所述扩增产物组装至质粒中,得到重组质粒;
A4:将所述重组质粒转移至大肠杆菌中后,在35~40℃下继续培养10~18h,对细菌进行筛选,得到初次筛选细菌;
A5:将所述初次筛选细菌接种于培养基中,并在35~40℃下继续培养12~18h,分离得到表达质粒;
A6:将所述表达质粒转移至枯草芽孢杆菌中,继续孵育2~3h后,在35~40℃下过夜培养,对细菌进行筛选,得到二次筛选细菌;
A7:将所述二次筛选细菌在摇瓶中培养,直到所述二次筛选细菌的细胞密度OD600达到0.6~0.8,得到摇瓶培养细菌;
A8:在所述摇瓶培养细菌中加入诱导剂诱导蛋白表达,25~30℃下继续培养15~20h,离心,得到含有纳米抗体的细菌上清液;或,使用所述摇瓶培养细菌制备芽孢,保存备用。
本发明中,所述纳米抗体具体可为抗咖啡因(Caffeine)纳米抗体、抗甲氨蝶呤(MTX)纳米抗体、抗小鼠细胞毒性T淋巴细胞相关蛋白4(CTLA-4)纳米抗体、抗小鼠程序性死亡配体1(PD-L1)纳米抗体中的任一种。
优选的,所述融合基因包括纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和碳水化合物结合结构域(CBM),从而得到含碳水化合物结合结构域(CBM)的融合蛋白。
优选的,所述枯草芽孢杆菌为WB800N感受态菌株。
优选的,所述大肠杆菌为DH5a菌株。
其中,所述WB800N感受态菌株的制备方法为:在使用前一天将枯草芽孢杆菌WB800N接种至培养基中,所述培养基包括质量浓度为1~1.1%磷酸氢钾、0.4~0.6%磷酸二氢钾、1~3%葡萄糖、0.05~0.1%柠檬酸钠二水合物、0.05~0.15%柠檬酸铁铵、0.2~0.3%天冬氨酸钾盐、0.03~0.06%酵母提取物和8~12mmol/L硫酸镁。
优选的,在步骤A7和A8中,所述摇瓶使用的培养介质为LB培养基,培养温度35~40℃,转速为200~300rpm,所述LB培养基包括质量分数为1%的胰蛋白酶原、1%的酵母抽提物和0.5%的NaCl。
优选的,步骤A8中使用的诱导剂为异丙基硫代半乳糖苷(IPTG)。
优选的,在步骤A8中,所述制备芽孢具体包括以下步骤:
B1:将所述摇瓶培养细菌涂布在琼脂板上,在35~40℃下孵育2天后,得到未纯化的芽孢,并将所述未纯化的芽孢从琼脂板上刮下;
B2:将所述未纯化的芽孢悬浮于冷水中,离心、弃上清液;
B3:将B2中的操作再重复2次,得到初步纯化的芽孢,再将所述初步纯化的芽孢重悬于冷水中,4℃下过夜;
B5:将B2~B3中的操作再重复2次,最终获得芽孢,所述芽孢被重悬于冷水中,在4℃下保存。
优选的,所述含碳水化合物结合结构域(CBM)的融合蛋白可以固定在碳水化合物上,用以长期保存和捕获小分子。
优选的,所述碳水化合物为再生无定形纤维素(RAC)。
优选的,所述再生无定形纤维素(RAC)的制备具体包括以下步骤:
C1:取微晶纤维素粉末加入双蒸水中,制成浆料;
C2:在所述浆料中加入冰磷酸,冰上孵育2h,随后再加入碱剂,将pH调至6.8~7.2;
C3:透析,得到所述再生无定形纤维素(RAC),冻干备用。
本发明的有益效果是:
首先,采用改良枯草芽孢杆菌菌株WB800N,其缺失8种胞外蛋白酶,可避免因枯草芽孢杆菌在培养上清中分泌蛋白酶,而导致分泌蛋白产量和质量显著降低的问题。
其次,构建的融合基因:该融合基因由纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和用于在含三乙酸(NTA)的层析柱上进行金属亲和层析的6×组氨酸标签(His标签)组成。也可使用碳水化合物结合结构域(CBM)取代His标签,如使用来自嗜热厌氧梭菌cipA基因的耐热纤维素结合域(CBD)取代His标签,以实现纤维素基融合蛋白的固定化或亲和纯化,其中:
CBD-纳米抗体融合蛋白的固定:该融合蛋白可固定于纤维素上,用于生产纤维素纸基生物传感或生物存储材料;
亲和纯化:将固定有该融合蛋白的纤维素,作为亲和色谱中的固定相,可用于捕获相应的小分子。如将分泌的CBD-咖啡因纳米抗体融合蛋白固定在纤维素上,并装载至注射器中,即得到了一个简单的色谱系统,可用于捕获咖啡因。通过更换纳米抗体的种类,即可用于其他物质的纯化。
本发明使用的载体质粒中含枯草芽孢杆菌来源groE基因操纵子的强启动子,在此基础上,引入大肠杆菌源乳糖操纵基因转化为IPTG诱导启动子。启动子区下游为核糖体结合位点(RBS)和amyQ基因分泌信号序列。考虑到amyQ分泌信号序列的胞外蛋白转运依赖细菌Sec途径,因此转运蛋白须行展开蛋白反应,后者依赖于固定于细菌膜的蛋白转运分子机制。在该过程中,由于降解或错误折叠,常出现多数哺乳动物重组蛋白的分泌并未达到可检测水平的问题。而在本发明中,融合基因包含具有高度热稳定性和/或相对分子量较小的重组蛋白,即目标纳米抗体,在该组合下,枯草芽孢杆菌的Sec依赖性分泌途径中不存在上述问题。
总体上,根据本发明的方法,得到的基于工程化枯草芽孢杆菌的多功能稳定平台,来生产纳米抗体,具有以下优势:
1.产量较高:摇瓶培养模式下每升培养上清中可产生15~20mg纳米抗体,且可通过补料、分批培养等进一步提高纳米抗体的产量,扩大产能。
2.低成本,长保质期:分泌的含碳水化合物结合结构域(CBM)的纳米抗体具有耐热性较好,可固定于碳水化合物如纤维素等底物上,成本低,可长期保存,且可在极端条件(如高温、紫外线照射、酸碱环境)下检测小分子。目前已知的,主要是以大肠杆菌为表达平台,制备上述重组融合蛋白,该过程中,需经历使用溶菌酶分解细菌的步骤,而枯草芽孢杆菌中Sec依赖分泌途径则是在细胞外产生重组蛋白。此外,CBM体积小和热稳定性高的特性有助于实现枯草芽孢杆菌的快速复性并抵抗蛋白酶的降解。
3.可用于癌症免疫治疗领域:如抗PD-Ll和抗CTLA-4,可用于免疫检查点阻断(ICB),目前,大多数抗PD-Ll和抗CTLA-4纳米抗体多产自大肠杆菌细胞内,但长时间的细菌溶解和内毒素清除过程可能会大大增加生产成本,且该过程需依赖于一定的集成化生产设备,成本较高,而通过工程化的枯草芽孢杆菌来生产纳米抗体,则可简化工艺,表现出显著的成本优势,且制成芽孢之后可保持长期稳定保存;
4.可用于敏感诊断实际的开发。该纳米抗体对低百分比(<1%)的细胞也可实现靶向识别,也可直接使用所述含有纳米抗体的细菌上清液对细胞表面进行蛋白质检测。
5.可用于炎症治疗领域:如通过纳米抗体生成阻断TNF-α等炎性细胞因子表达;
6.可用于胃肠道疾病治疗领域:芽孢对酸性环境的耐受能力也显示出了枯草芽孢杆菌作为载体生产纳米抗体,以改善胃肠道疾病的应用前景。
7.具有与其他技术相结合的应用潜力:如通过Sortase介导蛋白连接实现纳米抗体与荧光染料或其他肽序列的共价共轭等。
附图说明
下面结合附图对本发明作进一步说明。
图1为工程化枯草芽孢杆菌产芽孢作为纳米抗体稳定平台的示意图;
图2为生产纳米抗体的工艺路线;
图3为变性聚丙烯酰胺凝胶电泳(SDS-PAGE)的检测结果;
图4为免疫印迹法(western blot)检测靶蛋白存在与否的实验结果;
图5为CBD-抗咖啡因纳米抗体的分泌动力学检测实验结果(SDS-PAGE);
图6为CBD-抗咖啡因纳米抗体的分泌动力学检测实验结果(western blot);
图7为CBD-抗咖啡因纳米抗体的稳定性检测实验;
图8为CBD-抗咖啡因纳米抗体的特异性靶点识别验证实验;
图9为抗PD-L1纳米抗体与DC-2.4细胞结合情况的流式细胞仪检测结果;
图10为抗CTLA-4纳米抗体对低百分比细胞的靶向识别检测结果;
图11为对本发明所得的芽孢进行耐热性试验之后的检测结果;
图12为对本发明所得的芽孢进行耐酸性试验之后的检测结果。
具体实施方式
下面结合具体实施方式对本发明做进一步的说明。
材料和方法
化学用品:
除另有说明,本实验所有化学用品和细胞培养基均购自美国飞世尔科学世界公司(剑桥,马萨诸塞州,美国),且为商用最高纯度或分析级别。
Gibson Assembly Master Mix试剂盒和克隆酶购自新英格兰生物实验室公司(NEB)(伊普斯维奇,马萨诸塞州,美国)。DNA寡核苷酸由西格玛奥德里奇公司(圣路易斯,密苏里州,美国)设计和提供。质粒提取、琼脂糖凝胶DNA纯化和PCR清洁试剂盒购自CoWinBiosciences(剑桥,马萨诸塞州,美国)。DC蛋白分析试剂盒(#500-0116)购自Bio-Rad生命医学产品公司(赫尔克里斯,加利福尼亚州,美国)。
抗小鼠PD-L1和抗小鼠CTLA-4的纳米抗体序列来自RCSB蛋白结构数据库(PDB),并由Twist Bioscience公司(旧金山,加利福尼亚州,美国)合成基因片段。编码抗-MTX和抗咖啡因纳米抗体的大肠杆菌表达载体由北伊利诺伊大学的James Horn博士赠送。
质粒和菌株:
质粒和枯草芽孢杆菌WB800N菌株为越南国立大学科学院博士(越南国立大学)慷慨赠送,其中质粒已经过BamHI和XbaI酶切。将枯草芽孢杆菌WB800N置于Luria Bertani(LB,1%胰蛋白酶原、1%酵母抽提物和0.5%NaCl)培养基中培养。以氯霉素和新霉素作为筛选抗生素,其浓度分别为5μg/ml和10μg/ml。利用大肠杆菌DH5a菌株进行质粒构建和增殖。
构建融合基因中所用的引物:
注:大写字母代表是与目标基因互补的序列,小写字母则是与质粒骨架重叠的序列。
制备枯草芽孢杆菌WB800N感受态细胞:
步骤1:在使用前一天将枯草芽孢杆菌WB800N接种至培养基中,所述培养基包括质量浓度为1~1.1%磷酸氢钾、0.4~0.6%磷酸二氢钾、1~3%葡萄糖、0.05~0.1%柠檬酸钠二水合物、0.05~0.15%柠檬酸铁铵、0.2~0.3%天冬氨酸钾盐、0.03~0.06%酵母提取物和8~12mmol/L硫酸镁;
步骤2:在37℃下摇动,直到培养基中枯草芽孢杆菌WB800N的细胞密度OD600为1.0。
制备再生无定形纤维素(RAC):
步骤1:取1g平均粒径为50pm的微晶纤维素粉末加入3ml的双蒸水中,制成浆料;
步骤2:缓慢加入12.5ml的85%冰磷酸,冰上孵育2h;
步骤3:加入12.5g的固体氢氧化钠,将pH调至7;
步骤4:将样品移至截留分子量为3500的透析管中,加入4L双蒸馏水,透析48h,每6h更换一次双蒸馏水;
步骤5:将最后透析得到的RAC样品冻干备用。
实施例1:
步骤1:构建融合基因:从RCSB蛋白结构数据库(PDB)取得抗小鼠PD-L1和抗小鼠CTLA-4的纳米抗体序列,合成包含纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和用于在含三乙酸(NTA)的层析柱上进行金属亲和层析的6×组氨酸标签(His标签)的DNA片段,并对编码所述DNA片段的密码子进行优化;
步骤2:采用Q5高保真聚合酶扩增DNA片段,经Gibson组装、凝胶纯化,得到扩增产物;
步骤3:将所述扩增产物连接至经BamHI和XbaI酶切的质粒中,得到重组质粒;
步骤4:将所述重组质粒转移至大肠杆菌中后,在37℃下继续培养14h,对细菌进行筛选,得到初次筛选细菌;
步骤5:将所述初次筛选细菌接种于2ml的LB培养基中,并在37℃下培养15h后,分离得到表达质粒,并采用Sanger测序进行序列验证;
步骤6:将所述表达质粒转移至枯草芽孢杆菌WB800N感受态细胞中,继续孵育2.5h后,将100μl的培养液铺在琼脂板上,37℃下过夜培养,对细菌进行筛选,得到二次筛选细菌;对其中的菌落进行检测,验证是否产生了合适的重组蛋白;
步骤7:将所述二次筛选细菌在摇瓶中培养,培养介质为LB培养基,培养温度为37℃,转速为250rpm,直到所述二次筛选细菌的细胞密度OD600达到0.6~0.8,得到摇瓶培养细菌;
步骤8:在所述摇瓶培养细菌中加入1mmol/L的诱导剂IPTG诱导蛋白表达,28℃下继续培养18h,室温下高速离心(6000×g)30min,得到含有纳米抗体的细菌上清液。
实施例2:
步骤2-8与实施例1相同,步骤1如下:
构建融合基因:从RCSB蛋白结构数据库(PDB)取得抗咖啡因(Caffeine)、抗甲氨蝶呤(MTX)的纳米抗体序列,合成包含纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和取自嗜热厌氧梭菌cipA基因的耐热纤维素结合域(CBD)的DNA片段,并对编码所述DNA片段的密码子进行优化。
实施例3:
步骤1-7与实施例1相同,步骤8如下:
使用所述摇瓶培养细菌制备芽孢,保存备用,具体制备步骤为:
将所述摇瓶培养细菌涂布在琼脂板上,在37℃下孵育2天后,得到未纯化的芽孢,并将所述未纯化的芽孢从琼脂板上刮下,悬浮于冷水中,室温下高速离心(10000×g)10min、弃上清液;将重悬、离心、弃上清的操作再重复2次,得到初步纯化的芽孢,再将所述初步纯化的芽孢重悬于冷水中,4℃下过夜;再将上述操作重复2次,最终获得芽孢将所述芽孢被重悬于冷水中,在4℃下保存。
实施例4:
步骤1-7与实施例2相同,步骤8与实施例3相同。
进一步的说明参见附图。
图1为工程化枯草芽孢杆菌产芽孢作为纳米抗体稳定平台的示意图。含纳米抗体融合基因的芽孢在一定程度上可耐受多种极端环境,如辐射、紫外线、干燥、极端温度和温度波动以及酸性环境等。
图2为纳米抗体的生产流程。纳米抗体可与纤维素结合结构域(CBD)融合促进纤维素基的固定化,或与His标签融合以实现金属亲和纯化。枯草芽孢杆菌在长期贮存过程中会被诱导形成芽孢,为诱导纳米抗体的生成,将芽孢接种于培养基中,促进目标纳米抗体在Sec依赖蛋白分泌途径中的萌发、生长和分泌。其中,amyQ为蛋白分泌信号肽;groE是一种强启动子,由枯草杆菌groE操纵子衍生,通过引入大肠杆菌lac操纵子而转化为IPTG诱导启动子;GOI为感兴趣的基因(Gene of Interest),如纳米抗体的基因。考虑到amyQ分泌信号序列的胞外蛋白转运依赖细菌Sec途径,因此可能会由于降解或错误折叠,影响蛋白分泌的产量,事实上,Sec依赖分泌途径中,多数哺乳动物重组蛋白的分泌并未达到可检测水平。本发明分泌的蛋白为具有高度热稳定性和/或相对小分子量的含纳米抗体的重组蛋白,在枯草芽孢杆菌的Sec依赖性分泌途径中,并未发生上述不良结果。
对所得产品进行检测,结果参见图3~图12。
参见图3,发明人对四种不同纳米抗体进行了SDS-PAGE检测:(1)抗咖啡因;(2)抗甲氨蝶呤(MTX);(3)抗小鼠细胞毒性T淋巴细胞相关蛋白4(CTLA-4);(4)抗小鼠程序性死亡配体1(PD-L1)。前两种纳米抗体结合小分子在小分子检测方面具有潜在应用价值;后两种纳米抗体可有助于检测免疫细胞和肿瘤细胞表面抗原,具有潜在临床诊断价值,如提示患者对免疫检查点阻断疗法的反应。值得注意的是,与空载体质粒转化细菌相比,这四种纳米抗体在IPTG诱导6h后的细菌培养上清中都很容易能被检测到。
为确认SDS-PAGE凝胶上检测到的蛋白条带是否为期望得到的目标蛋白,发明人进一步使用融合蛋白克隆的FLAG表位标签的特异性抗体进行western blot检测。SDS-PAGE融合蛋白迁移位置为基于其计算所得分子量的预期位置。重组蛋白与FLAG表位融合后,应用抗FLAG抗体检测。参见图4,星号表示目标蛋白条带。
胞外表达无需细菌裂解步骤进行蛋白提取。但Sec依赖分泌途径中蛋白展开和再折叠步骤不可缺少,因此胞外表达产生速率可能相对慢于胞内表达速率。为了解诱导剂IPTG作用下枯草芽孢杆菌Nb分泌动力学特征,本实验采集不同时间点细菌培养上清供后续试验。
采用1mmol/L的IPTG诱导细菌生长。随后于各指定时间点采集0.5ml培养上清,应用三氯乙酸(TCA)沉淀蛋白,进行SDS-PAGE检测。参见图5,可知实验中检测到了CBD-抗咖啡因的纳米抗体,且其水平升高状态持续4h。重组蛋白(分子量大约为34kDa)在IPTG诱导后30min即可被检测到,矩形框表示在非诱导培养中无表达的目标蛋白。可以看出,当需要使用工程化的枯草芽孢杆菌生产所需纳米抗体时,其可在1h内分泌,这一特性有助于实现纳米抗体的按需生产。
图6为使用western blot方法对CBD-抗咖啡因纳米抗体的分泌动力学的检测结果,结论同上。
进一步地,发明人将含CBD-抗咖啡因纳米抗体的上清液滴加于滤纸上并干燥,放置3个月后进行免疫印迹检测,检测结果参见图7,该融合CBD的纳米抗体仍可被检测到。可见,CBD-抗咖啡因纳米抗体可固定于纤维素上,且稳定性良好,3个月后仍保持较好的状态,其在纤维素纸基生物传感或生物存储方面具有潜在的应用前景。
在进行本发明所得纳米抗体的特异性靶点识别验证实验过程中,发明人按照以下步骤设计了一个色谱柱:
步骤1:装载色谱柱。将0.1ml再生无定形纤维素(RAC)(50mg干重)装载入1ml注射器中。其中,固定相是上述采用85%磷酸水解纤维素微晶制备的RAC,水解后的纤维素具有更大比表面积,有利于实现其与CBD的最大化结合;150ug/ml的咖啡因溶液为流动相,该溶液是在磷酸盐缓冲液(PBS)中制备而成的。
步骤2:将含有CBD-抗咖啡因纳米抗体(CBD-抗MTX纳米抗体为阴性对照组)的细菌上清液上样至所述色谱柱,直至饱和;其中,采用SDS-PAGE检测CBD-抗咖啡因纳米抗体(或CBD-抗MTX纳米抗体)的存在,以确定色谱柱是否达到饱和状态;
步骤3:流动相连续通过所述含有饱和纳米抗体的色谱柱。依通过固定相的时间顺序收集5组流动相,每组100微升,并对采用紫外分光光度计分析各组流动相中残留的咖啡因的浓度。
参加图8可得结果如下:与CBD-抗MTX组相比,流动相通过含有CBD-抗咖啡因纳米抗体的RAC柱时,流动相中的咖啡因水平显著降低,从而证明了分泌的CBD-抗咖啡因纳米抗体对特定靶点的特异性识别功能。继续参见图8可知,大约每500g含有CBD-抗咖啡因纳米抗体的固定相可与10g咖啡因相结合,这种结合的咖啡因的量与相对应的融合抗体具有一一对应的关系。这种定量的结果可用于指导制备具有靶向、定量结合的亲和纯化色谱系统。
随后采用枯草芽孢杆菌分泌的纳米抗体检测蛋白抗原,验证对应纳米抗体得靶向识别功能。
发明人采用纯化后的抗PD-Ll纳米抗体、抗咖啡因纳米抗体(阴性对照组)、未经纯化的含有抗PD-Ll纳米抗体的细菌上清液、未经纯化的含有抗咖啡因纳米抗体(阴性对照组)的细菌上清液对DC-2.4细胞进行免疫染色,采用流式细胞仪分析染色样品;其中,DC2.4细胞为小鼠树突状细胞,细胞表面具有丰富的抗原,本实验中该细胞表面的抗原为免疫抑制受体PD-L1。
参见图9,流式细胞仪检测结果表明,与未染色的样品相比,经纯化抗PD-L1纳米抗体具有特异性结合能力,而抗咖啡因纳米抗体对照组未显示出相应结合能力。同时,未经纯化的含有PD-L1特异性纳米抗体的细菌上清中检测有PD-L1表达,而其在抗咖啡因纳米抗体对照组中无表达。
图10为抗CTLA-4纳米抗体对低百分比细胞的靶向识别检测结果。在低百分比细胞的检测中,细胞中的CTLA-4仍有阳性表达,表现出开发敏感诊断试剂的应用前景。
对于实施例3和实施例4得到的芽孢进行抗性试验检测。
耐热性试验步骤如下:
步骤1:将芽孢和营养细胞滴加至惠特曼滤纸上,暴露于波士顿(东北大学ISEC大楼的花园区)105°F阳光下,分别持续24h和48h;
步骤2:回收所有热暴露后滤纸样本,使用新鲜LB培养基浸润滤纸,并置于含有所需抗生素的LB培养基平皿上,37℃孵育过夜,对形成的菌落进行计数;
步骤3:将经热处理过的芽孢接种至生长培养基中,其能够产生与未处理菌株相当水平的抗CTLA-4纳米抗体。
参见图11,休眠状态的枯草芽孢杆菌,即芽孢可耐受环境热和阳光照射的综合影响,并能够从热暴露中恢复,而营养细胞则未表现出类似的特性。图上箭头表示在滴加有芽孢的滤纸上的菌落生长状况,而营养细胞无菌落生长。此外,上述经过耐热试验的芽孢仍然具备生产抗CTLA-4纳米抗体的能力,且无明显的产量损失(如图11中的方框所示)。
耐酸性试验步骤如下:
步骤1:将芽孢和营养细胞样本稀释至OD600=0.01,取5μl样品分别置于pH=1、pH=2.9的含有盐酸的0.9%(wt/wt)氯化钠溶液中,并处理30min、1h和2h;
步骤2:将酸处理过的样品置于含有所需抗生素的LB培养基平皿上,37℃孵育过夜,对形成的菌落进行计数;
步骤3:将经酸处理过的芽孢接种至生长培养基中,其能够产生与未处理菌株相当水平的抗CTLA-4纳米抗体。
参见图12,休眠状态的枯草芽孢杆菌,即芽孢可耐受模拟胃液pH酸性环境的酸性pH(pH=1和pH=2.9)刺激,生长状况良好,而营养细胞不具备此耐受性,并无菌落生长。此外,上述经过酸处理的芽孢仍然具备生产抗CTLA-4纳米抗体的能力,且无明显的产量损失(如图12中的方框所示)。
芽孢对酸性环境的耐受能力也显示出了枯草芽孢杆菌作为载体生产纳米抗体,以改善胃肠道疾病的应用前景。
除上述优选实施例外,本发明还有其他的实施方式,本领域技术人员可以根据本发明作出各种改变和变形,只要不脱离本发明的精神,均应属于本发明权利要求书中所定义的范围。
Claims (10)
1.一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:包括以下步骤:
A1:构建融合基因:所述融合基因包括纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和用于在含三乙酸(NTA)的层析柱上进行金属亲和层析的6×组氨酸标签(His标签);合成DNA片段,并对编码所述DNA片段的密码子进行优化;
A2:扩增所述DNA片段,并纯化,得到扩增产物;
A3:将所述扩增产物组装至质粒中,得到重组质粒;
A4:将所述重组质粒转移至大肠杆菌中后,在35~40℃下继续培养10~18h,对细菌进行筛选,得到初次筛选细菌;
A5:将所述初次筛选细菌接种于培养基中,并在35~40℃下继续培养12~18h,分离得到表达质粒;
A6:将所述表达质粒转移至枯草芽孢杆菌中,继续孵育2~3h后,在35~40℃下过夜培养,对细菌进行筛选,得到二次筛选细菌;
A7:将所述二次筛选细菌在摇瓶中培养,直到所述二次筛选细菌的细胞密度OD600达到0.6~0.8,得到摇瓶培养细菌;
A8:在所述摇瓶培养细菌中加入诱导剂诱导蛋白表达,25~30℃下继续培养15~20h,离心,得到含有纳米抗体的细菌上清液;或,使用所述摇瓶培养细菌制备芽孢,保存备用。
2.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述纳米抗体为抗咖啡因(Caffeine)纳米抗体、抗甲氨蝶呤(MTX)纳米抗体、抗小鼠细胞毒性T淋巴细胞相关蛋白4(CTLA-4)纳米抗体、抗小鼠程序性死亡配体1(PD-L1)纳米抗体中的任一种。
3.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述融合基因包括纳米抗体的DNA序列、用于融合蛋白检测的FLAG肽(DYKDDDDK)和碳水化合物结合结构域(CBM),从而得到含碳水化合物结合结构域(CBM)的融合蛋白。
4.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述枯草芽孢杆菌为WB800N感受态菌株。
5.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述大肠杆菌为DH5a菌株。
6.根据权利要求4所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述WB800N感受态菌株的制备方法为:在使用前一天将枯草芽孢杆菌WB800N接种至培养基中,所述培养基包括质量浓度为1~1.1%磷酸氢钾、0.4~0.6%磷酸二氢钾、1~3%葡萄糖、0.05~0.1%柠檬酸钠二水合物、0.05~0.15%柠檬酸铁铵、0.2~0.3%天冬氨酸钾盐、0.03~0.06%酵母提取物和8~12mmol/L硫酸镁。
7.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述摇瓶中使用的培养介质为LB培养基,培养温度35~40℃,转速为200~300rpm,所述LB培养基包括质量分数为1%的胰蛋白酶原、1%的酵母抽提物和0.5%的NaCl;和/或,所述诱导剂为异丙基硫代半乳糖苷(IPTG)。
8.根据权利要求1所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述制备芽孢具体包括以下步骤:
B1:将所述摇瓶培养细菌涂布在琼脂板上,在35~40℃下孵育2天后,得到未纯化的芽孢,并将所述未纯化的芽孢从琼脂板上刮下;
B2:将所述未纯化的芽孢悬浮于冷水中,离心、弃上清液;
B3:将B2中的操作再重复2次,得到初步纯化的芽孢,再将所述初步纯化的芽孢重悬于冷水中,4℃下过夜;
B5:将B2~B3中的操作再重复2次,最终获得芽孢,所述芽孢被重悬于冷水中,在4℃下保存。
9.根据权利要求3所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述含碳水化合物结合结构域(CBM)的融合蛋白可以固定在碳水化合物上,用以长期保存和捕获小分子。
10.根据权利要求9所述的一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,其特征在于:所述碳水化合物为再生无定形纤维素(RAC);和/或,所述再生无定形纤维素(RAC)的制备具体包括以下步骤:
C1:取微晶纤维素粉末加入双蒸水中,制成浆料;
C2:在所述浆料中加入冰磷酸,冰上孵育2h,随后再加入碱剂,将pH调至6.8~7.2;
C3:透析,得到所述再生无定形纤维素(RAC),冻干备用。
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WO2021153129A1 (ja) * | 2020-01-27 | 2021-08-05 | 花王株式会社 | 重鎖抗体の重鎖可変ドメインの製造方法 |
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