CN114891817A - 一种多聚肽及其制备方法与应用 - Google Patents
一种多聚肽及其制备方法与应用 Download PDFInfo
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- CN114891817A CN114891817A CN202210397319.7A CN202210397319A CN114891817A CN 114891817 A CN114891817 A CN 114891817A CN 202210397319 A CN202210397319 A CN 202210397319A CN 114891817 A CN114891817 A CN 114891817A
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Abstract
本发明公开了一种多聚肽及其制备方法与应用,属于基因工程领域,特别是蛋白或多肽类药物的制备,具体涉及一种多聚抗病毒肽的制备及应用。具体方法:由一个与SpyTag肽融合的目标多肽,及一个SpyCatcher肽和类弹性蛋白ELP肽融合的骨架蛋白;目标多肽和骨架蛋白无需柱纯化、通过SpyCatcher/Tag高效自连接即可一步获得多聚肽,大大提高产率。该方法可应用于提高目标多肽的亲和力、稳定性等重要生物学属性。
Description
技术领域
本发明公开了一种多聚肽的制备方法及应用,属于基因工程领域,特别是蛋白或多肽类药物的制备,具体涉及一种多聚抗病毒肽的制备及应用。
背景技术
严重急性呼吸系统综合征2型冠状病毒((severe acute respiratory syndromecoronavirus 2,SARS-CoV-2))导致的传染性肺炎,无论是在感染人数和流行区域上,都远远超过了前两次的SARS和MERS,世界卫生组织(WHO)紧急宣布将新型冠状病毒肺炎疫情列为国际关注的突发公共卫生事件(PHEIC)(Lai CC,2020)。
疫苗叠加治疗药物,成为新冠病毒防治新策略。然而,尽管疫苗在全球接种加速,但是治疗药物依是不可或缺的一环。目前,治疗新型冠状病毒肺炎的药物主要包括广谱抗病毒药物、S蛋白抑制剂、ACE2抑制剂、RdRp抑制剂、3CLpro和PLpro 抑制剂等。新冠肺炎疫情下,多个国家和地区都对新冠用药加快了审批速度,目前已获批的药物包括3个小分子药物,7个中和抗体类药物。辉瑞公司的小分子口服药Paxlovid已经在《新型冠状病毒肺炎诊疗方案(试行第九版)》中被推荐使用。但以新冠病毒的变异和传播速度,要成功抗击疫情,还是需要研发更多不同作用机制的抗新冠病毒药物。
根据SARS-CoV-2的RNA基因组结构,目前对抗病毒新药的研发主要集中于两个靶点(V'kovski P等,2021):(1)非结构性蛋白(Nonstructural proteins,NSPs):病毒编码的蛋白多聚体pp1a和pp1ab被木瓜蛋白酶样蛋白酶(Papain-like proteinase, PLpro)和3-胰凝乳蛋白酶样蛋白酶(Main proteinase 3CL,Mpro或3CLpro)水解后产生多种复制相关的非结构蛋白酶,抑制这些酶可以阻止SARS-CoV-2的复制。(2)刺突蛋白(Spike,S):刺突蛋白和宿主细胞上的血管紧张素2(Angiotensin converting enzyme 2,ACE2)结合,相互作用促进细胞膜融合入侵体内。
科学家们通过靶向S蛋白筛选了多种能够阻断SARS-CoV-2与ACE2结合的化学药物,以及新的抗体和小分子抗病毒肽。抗体和小分子抗病毒肽是具有巨大前景的生物药物,它们能够识别特定的蛋白结构并与其结合达到抑制蛋白活性的目的,常用于恶性肿瘤、自身免疫病。小分子肽由于其分子小,可以更好地结合更广泛的胞内和胞外靶点(Bedard P L等,2020)。但筛选出来的小分子肽距离成药还面临诸多难题,如免疫原性、脱靶效应等。除此之外,小分子肽还面临着一个难题,即半衰期短、稳定性低。
针对这个问题,科学家摸索出了多种解决方式,常用聚乙二醇(PEG)修饰(Magdalena Swierczewska等,2015)、用白蛋白(HSA)修饰小分子肽(Dennis M S,2002)。但PEG化修饰也存在局限性,比如PEG无法在体内降解;HSA融合的瓶颈问题是可能会影响目的蛋白质的正确折叠,HSA的空间位阻也会目的蛋白发挥生物学活性。
类弹性蛋白ELP(Elastin-Like Polymers)是一种由五肽重复单元(Val-Pro-Gly-Xaa-Gly,VPGXG)串联而成的新兴的药物递送材料,其中,Xaa是除去脯氨酸外的任意氨基酸。ELP肽作为一种以天然氨基酸为基元的生物大分子,能生物降解成身体所需的氨基酸,ELP肽可以作为一个不错的生物材料用来改善药物特性,延长药物半衰期,降低药物副作用。
结合ELP肽多聚结合可以延长半衰期,增加稳定性,除此之外,多聚结合还具有多种优点(Carlescu I,2008):其一,多聚抗病毒肽通过与病原体多靶位的结合,可以降低病原体逃逸突变产生耐药性的可能;其二,多聚小分子抗病毒肽相较于单个小分子,能显著提高靶向亲和力,提高选择性。
然而目前没有高效的多聚抗病毒肽制备方法,严重影响了多聚抗病毒肽的研发与应用。
发明内容
为了克服现有技术存在的不足,本发明的目的是提供一种高亲和力多聚抗病毒肽的制备方法及应用。
本发明提供了一种基于Spy化学,结合类弹性蛋白ELP的特性,制备了一种靶向SARS-Cov-2病毒的高亲和力多聚抗病毒肽及其应用。该方法是一种通过表达所述融合多肽来制备和纯化目的多聚抗病毒肽的方法,所述融合多肽包含C端与 SpyTag肽融合的抗病毒肽LCB3单体和融合多个SpyCatcher肽和ELP肽的骨架蛋白。
本发明提供的高亲和力多聚抗病毒肽的制备方法,是通过分子克隆构建C端与SpyTag肽融合的抗病毒肽LCB3单体的融合蛋白基因和融合多个SpyCatcher肽和 ELP肽和骨架蛋白,然后分别导入宿主细胞中,进行培养宿主细胞,以表达所述融合蛋白,接着裂解宿主细胞,将C端与SpyTag融合的抗病毒肽LCB3单体的融合蛋白和融合多个SpyCatcher肽和ELP肽的骨架蛋白混合,通过进一步的金属亲和层析和尺寸排阻色谱纯化,得到二聚或者三聚LCB3抗病毒肽。
一种高亲和力多聚抗病毒肽的制备方法,包括如下步骤:
—构建C端与SpyTag肽融合的抗病毒肽LCB3单体;
—构建融合多个SpyCatcher肽和ELP肽的骨架蛋白;
—混合融合SpyCatcher肽和ELP肽的骨架蛋白与融合有SpyTag肽的抗病毒肽LCB3单体,使二者特异性共价结合,后通过两轮纯化获取多聚肽。
—多聚肽的生物膜层干涉实验和细胞中和实验证实多聚修饰提升的亲和力。
上述方法中,所述C端与SpyTag肽融合的抗病毒肽LCB3单体的制备方法包括:
(1)将聚集肽的基因序列、切割标签的基因序列、抗病毒肽LCB3、SpyTag 肽的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌;培养所述工程菌表达所述融合蛋白,然后裂解工程菌,离心取沉淀,得到融合蛋白的聚集体;将所述融合蛋白的聚集体进行切割,得到C端与SpyTag 肽融合的抗病毒肽LCB3单体。
(2)将抗病毒肽LCB3和SpyTag肽的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞表达,得到工程菌,超声裂解,得到融合蛋白的溶液。上述方法中,方法(1)中,所述两亲性自组装短肽为类表面活性剂短肽;所述类表面活性剂短肽的氨基酸序列如SEQ ID NO:10所示。
上述方法中,方法(1)中,所述切割标签为化学切割位点、酶法切割位点或自切割位点。
上述方法中,方法(1)中,所述切割标签为自切割位点,所述自切割位点为内含肽。
上述方法中,方法(1)中,所述内含肽为MtuΔI-CM(WT),优选地,所述 MtuΔI-CM(WT)的氨基酸序列如SEQ ID NO:11所示。
上述方法中,方法(1)中,所述内含肽为MtuΔI-CM的突变体,所述MtuΔ I-CM的突变体为MtuΔI-CM的m1、m2或m3突变体,所述MtuΔI-CM的m1 突变体的氨基酸序列如SEQ IDNO:12所示,所述MtuΔI-CM的m2突变体的氨基酸序列如SEQ ID NO:13所示,所述MtuΔI-CM的m3突变体的氨基酸序列如SEQ ID NO:14所示。
上述方法中,当所述自切割位点为内含肽时,步骤(3)中所述切割,包括:将所述融合蛋白的聚集体分散在缓冲液中,进行切割处理,离心取上清液,得到含C 端融合SpyTag肽的抗病毒肽LCB3的溶液;所述缓冲液的pH值为5.5~6.8,所述切割处理的温度为4~37℃,切割处理的时间为3~48h。
上述方法中,优选地,所述SpyTag肽的氨基酸序列如SEQ ID NO:6所示,LCB3 肽的氨基酸序列如SEQ ID NO:1所示。
上述方法中,所述聚集肽的基因序列与切割标签的基因序列之间通过接头连接,所述接头为PT型接头;所述PT型接头的氨基酸序列如SEQ ID NO:9所示。
上述方法中,所述融合SpyCatcher的个数为两个或三个,所述融合ELP肽单元的个数为一个或两个。所述的SpyCatcher氨基酸序列如SEQ ID NO:3所示,所述的 ELP肽氨基酸序列如SEQ ID NO:2所示。
所述融合多个SpyCatcher肽和ELP肽的骨架蛋白的制备方法:将ELP肽和SpyCatcher肽的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞表达,得到工程菌,超声裂解,得到骨架蛋白的溶液。
上述方法中,将融合SpyCatcher肽和ELP肽的骨架蛋白与融合有SpyTag肽的抗病毒肽LCB3单体按照SpyCatcher肽:SpyTag肽的摩尔比接近1:1的数量关系进行投料,融合有SpyTag的抗病毒肽LCB3单体过量的情况下,可以通过金属亲和层析将二聚LCB3抗病毒肽和三聚LCB3抗病毒肽进行纯化,再通过进一步的尺寸排阻色谱法的精细纯化,得到高纯度的二聚LCB3抗病毒肽和三聚LCB3抗病毒肽。
本发明提供了一种多核苷酸,其包含编码本发明上述的融合蛋白的核苷酸序列或其互补序列。
本发明提供了一种表达载构建体,其包含本发明上述的多核苷酸。
本发明提供了一种宿主细胞,其包含本发明上述的多核苷酸或以本发明所述的表达载构建体,其中所述宿主细胞能够表达本发明上述的融合蛋白。
在本发明的重组表达构建体中,编码所述融合蛋白的多核苷酸序列与表达控制序列进行适当连接以实现如期的转录及最终在宿主细胞中生产所述融合蛋白。所述的表达控制序列包括但不限于启动子、增强子、核糖体结合位点、聚腺苷酸化位点、转录剪接序列、转录终止序列和稳定mRNA的序列等。
本发明用于表达构建体的载体包括可在宿主细胞中自主复制的载体,如质粒载体;和可整合到宿主细胞DNA中并和宿主细胞DNA一起复制的载体。在具体实施方案中,本发明所述的表达构建体衍生自Novagen公司的pET30a(+)和pET32a(+)。用于表达本发明融合蛋白的宿主细胞包括原核生物、酵母和高等真核细胞。示例性的原核生物包括埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)的细菌。在优选的实施方案中,宿主细胞是埃希氏菌属细胞,优选是大肠杆菌。在本发明的一个具体实施方案中,所使用的宿主细胞为大肠杆菌BL21(DE3)菌株细胞(Novagen)。
在本发明中,采用了C端与SpyTag肽融合的抗病毒肽LCB3单体和融合多个SpyCatcher肽和ELP肽的骨架蛋白进行多聚化,保留了抗病毒肽LCB3的N端完整,规避了N末端修饰造成的活性减弱,采用的ELP肽无毒性、可生物降解、具有良好的生物相容性,作为药物递送材料来使用非常安全可靠。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明通过SpyCatcher/Tag的高效、高特异性反应,骨架蛋白和目标蛋白无需柱纯化,即可快速反应获得多肽抗病毒肽。
(2)本发明通过利用在常温不聚集的ELP肽序列,避免目前ELP肽在医药领域应用时由于易聚集而产生毒性的问题。
(3)本发明所获多肽抗病毒肽在亲和力和抗病毒活性上有显著提高。
附图说明
图1A是cSAT法表达和纯化LCB3-SpyTag融合蛋白的示意图;
图1B是基于cSAT法LCB3-SpyTag融合蛋白表达载体的结构示意图;
图1C是直接表达的LCB3-SpyTag融合蛋白表达载体的结构示意图。
图2A是基于cSAT法采用MtuΔI-CM(WT)、MtuΔI-CM m1、MtuΔI-CM m2内含肽作为切割标签时的LCB3-SpyTag融合蛋白表达纯化产物的SDS-PAGE结果;
图2B是基于cSAT法采用MtuΔI-CM(WT)和MtuΔI-CM m1内含肽作为切割标签时的LCB3-SpyTag融合蛋白的表达纯化过程的SDS-PAGE结果;
图2C是基于cSAT法采用MtuΔI-CM m2内含肽作为切割标签时的LCB3-SpyTag 融合蛋白的表达纯化过程的SDS-PAGE结果;
图2D是基于cSAT法采用MtuΔI-CM m3内含肽作为切割标签时的LCB3-SpyTag 融合蛋白的表达纯化过程的SDS-PAGE结果;
图2E是基于cSAT法采用MtuΔI-CM m3内含肽作为切割标签时的LCB3-SpyTag融合蛋白表达纯化产物和LCB3-SpyTag直接表达的SDS-PAGE结果;
图3A是LCB3-HisTag融合蛋白表达载体的结构示意图;
图3B是融合两个SpyCatcher的ELP骨架蛋白表达载体示意图;
图3C是融合三个SpyCatcher的ELP骨架蛋白表达载体示意图;
图4A是融合两个SpyCatcher的ELP骨架蛋白表达的SDS-PAGE检测结果;
图4B为LCB3-HisTag融合蛋白、融合三个SpyCatcher的ELP骨架蛋白表达的 SDS-PAGE检测结果;
图5A是多聚LCB3抗新冠病毒肽的组装;
图5B是通过两轮纯化的样品最终的SDS-PAGE检测结果;
图6A是LCB3-HisTag融合蛋白与新冠病毒刺突蛋白RBD的BLI检测结果;
图6B是二聚LCB3抗新冠病毒肽与新冠病毒刺突蛋白RBD的BLI检测结果;
图6C是三聚LCB3抗新冠病毒肽与新冠病毒刺突蛋白RBD的BLI检测结果;
图7A是LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒中和实验检测结果;
图7B是LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的对含新冠病毒刺突蛋白delta变体假病毒中和实验检测结果。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明,但本发明的实施和保护不限于此。需指出的是,以下若有未特别详细说明之过程,均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者,视为可以通过市售购买得到的常规产品。
一种多聚肽的制备方法,包括如下步骤:
(a)构建及表达融合SpyTag肽的目标多肽;
(b)构建及表达融合SpyCatcher肽和ELP肽的骨架蛋白;将ELP肽和 SpyCatcher肽的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌,超声裂解,进行金属亲和层析和尺寸排阻色谱纯化分离;
(c)混合目标多肽和骨架蛋白,自连接形成多聚肽;
(d)纯化获得高纯度多聚肽。
上述方法中,所述的目标多肽是抗体、纳米抗体、蛋白、抗菌肽或抗病毒肽;优选抗病毒肽。
上述方法中,所述抗病毒肽是抗新型冠状病毒肽,优选LCB3多肽(SEQ ID NO:1)。
上述方法中,所述骨架蛋白的SpyCatcher肽和ELP肽以交替重复顺序融合,其重复顺序特征为:
(a)SpyCatcher肽的重复次数为n次,ELP肽的重复次数为n-1至n+1次;其中n大于或等于2,优选为2和3;
(b)A-B-A、A-B-A-B-A或B-A-B-A-B、B-A-B-A-B-A-B。
上述方法中,所述ELP肽的特征满足如下条件:
(a)Val-Pro-Gly-Xaa-Gly的重复序列;
(b)Val-Pro-Gly-Xaa-Gly重复序列的重复数为10~30次,优选12~18次;
(c)其中Xaa为任意氨基酸,优选为Val和Glu;
(d)其中Xaa氨基酸Val和Glu的比例3:1~5:1,优选4:1
(e)ELP肽为SEQ ID NO:2所示的氨基权序列。
上述方法中,SpyCatcher肽与所述的SpyTag肽形成异肽链,优选地,所述SpyCatcher肽包含选自SEQ ID NO:3,4,5任一所示的氨基酸序列,所述SpyTag 肽包含选自SEQ ID NO:6,7,8任一所示的氨基酸序列。
上述方法中,所述融合SpyTag肽的目标多肽的制备方法,包括如下步骤:
将目标多肽、SpyTag肽和纯化标签的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌;培养所述工程菌表达所述融合蛋白,然后裂解工程菌,捕获得到所述融合蛋白;
所述的纯化标签由聚集肽和切割标签组成;所述聚集肽的基因序列与切割标签的基因序列之间通过接头连接,所述接头为PT型接头;所述PT型接头为如SEQ ID NO:9所示氨基酸序列。
所述聚集肽为两亲性自组装短肽,优选为类表面活性剂短肽;所述类表面活性剂短肽为如SEQ ID NO:10所示氨基酸序列;
所述切割标签为化学切割位点、酶法切割位点或自切割位点,优选为自切割位点;所述自切割位点为内含肽,优选MtuΔI-CM内含肽或其突变体为如SEQ ID NO:11,12,13,14所示氨基酸序列;
所述捕获得到所述捕获,包括将工程菌裂解后离心取沉淀,得到融合蛋白的聚集体;将所述融合蛋白的聚集体进行切割。
所述切割,包括:将所述融合蛋白的聚集体分散在缓冲液中,进行切割处理,离心取上清液,得到所述融合蛋白的溶液;所述缓冲液的pH值为5.5~6.8,所述切割处理的温度为4~37℃,切割处理的时间为3~48h。
上述方法中,所述骨架蛋白的制备方法包括如下步骤:
(a)将SpyCatcher肽,ELP肽和纯化标签的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌;所述纯化标签为亲和纯化标签,优化为HisTag标签;
(b)所述工程菌表达所述融合蛋白,然后裂解工程菌,得到所述融合蛋白的溶液。
上述方法中,所述多聚肽的制备方法,包括如下步骤:
(a)将融合SpyTag肽的目标多肽和骨架蛋白,按各自融合蛋白所具有的SpyTag 和SpyCatcher的摩尔比的1~1.5倍进行混合反应,得到多聚肽溶液;所述混合反应的温度为4~37℃,混合反应的时间为3~24h;
(b)将多聚肽溶液,通过金属亲和层析和尺寸排阻色谱进行纯化,得到高纯度的多聚肽。
上述多聚肽为多聚抗新冠病毒肽,优选为二聚LCB3抗新冠病毒肽,三聚LCB3 抗新冠病毒肽;
所述二聚LCB3抗新冠病毒肽对新冠病毒刺突蛋白RBD的解离常数Kd小于1 pM,所述三聚LCB3抗新冠病毒肽对新冠病毒刺突蛋白RBD的解离常数Kd小于1 pM。
所述二聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒的半数抑制浓度 IC50为0.57nM,所述三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒的半数抑制浓度IC50为0.20nM。
所述二聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白delta变体假病毒的半数抑制浓度IC50为0.53nM,所述三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白delta 变体假病毒的半数抑制浓度IC50为0.32nM。
实施例1:构建含有和不含内含肽MtuΔI-CM不同突变株的C端与SpyTag融合的抗病毒肽LCB3(简称LCB3-SpyTag融合蛋白)表达载体
本申请实施例中所使用的表达载体为含4种不同MtuΔI-CM突变株的融合蛋白表达载体(pET32a-L6KD-PT-MtuΔI-CM(WT)-LCB3-SpyTag、pET32a-L6KD-PT-Mtu ΔI-CM m1-LCB3-SpyTag、pET32a-L6KD-PT-MtuΔI-CM m2-LCB3-SpyTag、 pET32a-L6KD-PT-MtuΔI-CMm3-LCB3-SpyTag),以及不含内含肽直接表达的融合蛋白表达载体(pET32a-LCB3-SpyTag)。图1A为cSAT法表达和纯化LCB3-SpyTag 融合蛋白的示意图;图1B为基于cSAT法LCB3-SpyTag融合蛋白表达载体的结构示意图;图1C为直接表达的LCB3-SpyTag融合蛋白表达载体的结构示意图。
首先构建pET32a-L6KD-PT-MtuΔI-CM(WT)-LCB3-SpyTag质粒,该质粒构建所需要的引物,通过oligo 7设计并由上海生工合成寡聚核苷酸引物。首先将抗病毒肽 LCB3的氨基酸序列(SEQ ID NO:1)进行大肠杆菌宿主密码子优化,优化后抗病毒肽LCB3基因由上海生工合成。通过引物(OY-20-004-F/OY-20-004-R,如SEQ ID NO:15/SEQ ID NO:16所示)进行PCR扩增得到目的LCB3基因片段,然后用引物(OY-20-006-F/OY-20-001-R和OY-20-002-F/OY-20-002-R,如SEQ ID NO:17/SEQ ID NO:18和SEQ ID NO:19/SEQ ID NO:20所示)对实验室原有的pET32a-L6KD-PT-Mtu ΔI-CM(WT)-VHH72-SpyTag进行分段扩增,得到含有LacI-L6KD-PT-MtuΔI-CM(WT)的基因片段和含有SpyTag-AmpR-Ori的基因片段,将含有SpyTag-AmpR-Ori的基因片段与扩增出的LCB3基因片段用引物(OY-20-004-F/OY-20-002-R,如SEQ ID NO:15/SEQ ID NO:20所示)通过重叠PCR的方法扩增出含有 LCB3-SpyTag-AmpR-Ori的基因片段,随后将含有LCB3-SpyTag-AmpR-Ori的基因片段和用含有Lac I-L6KD-PT-MtuΔI-CM(WT)的基因片段进行Gibson assembly,将获得的连接产物化转入大肠杆菌DH5α,通过菌落PCR鉴定和测序筛选出正确的阳性克隆后,提取该质粒转化大肠杆菌表达菌株BL21(DE3)进行蛋白表达以及后续实验。
pET32a-L6KD-PT-MtuΔI-CM m1-LCB3-SpyTag、pET32a-L6KD-PT-MtuΔI-CM m2-LCB3-SpyTag、pET32a-L6KD-PT-MtuΔI-CM m3-LCB3-SpyTag质粒以上述构建的pET30a-L6KD-PT-MtuΔI-CM(WT)-LCB3-SpyTag质粒为模板,通过Gibson assembly的方法构建。用引物通过PCR反应以pET32a-L6KD-PT-Mtu ΔI-CM(WT)-LCB3-SpyTag质粒为模板,利用引物(OY-20-004-F/OY-20-001-R,如 SEQ ID NO:15/SEQ ID NO:18所示)通过PCR反应扩增LCB3-SpyTag-AmpR-Ori片段,作为Gibson assembly的片段。以实验室原有的质粒pET32a-L6KD-PT-MtuΔI m1-CM-hGH、pET32a-L6KD-PT-MtuΔI-CM m2-hGH、pET32a-L6KD-PT-MtuΔI-CM m3-hGH为模板,利用引物(OY-20-002-F/OY-20-002-R,如SEQ ID NO:19/SEQ ID NO:20所示)通过PCR反应扩增L6KD-PT-MtuΔI-CM m1、L6KD-PT-MtuΔI-CM m2、 L6KD-PT-MtuΔI-CM m3片段,以LCB3-SpyTag-AmpR-Ori/Lac1-L6KD-PT-Mtu ΔI-CM m1、LCB3-SpyTag-AmpR-Ori/Lac1-L6KD-PT-MtuΔI-CM m2、 LCB3-SpyTag-AmpR-Ori/Lac1-L6KD-PT-MtuΔI-CM m3三对片段进行Gibson assembly。将获得的连接产物化转入大肠杆菌DH5α,通过菌落PCR鉴定和测序筛选出正确的阳性克隆后,提取该质粒转化大肠杆菌表达菌株BL21(DE3)进行表达以及后续实验。
pET32a-LCB3-SpyTag质粒以上述构建的pET30a-L6KD-PT-MtuΔI CM(WT)-LCB3-SpyTag质粒为模板,通过Gibson assembly的方法构建。利用引物(OY-21-002-F/OY-21-014-R,如SEQ ID NO:21/SEQ ID NO:24所示)通过PCR反应扩增LCB3-SpyTag-AmpR-Ori片段,利用引物(OY-21-014-F/OY-21-002-R,如SEQ ID NO:25/SEQ ID NO:26所示)通过PCR反应扩增Lac I片段,以 LCB3-SpyTag-AmpR-Ori/Lac I片段进行Gibson assembly。将获得的连接产物化转入大肠杆菌DH5α,通过菌落PCR鉴定和测序筛选出正确的阳性克隆后,提取该质粒转化大肠杆菌表达菌株BL21(DE3)进行表达以及后续实验。
实施例2:含有内含肽MtuΔI-CM不同突变株的LCB3-SpyTag的表达纯化和 LCB3-SpyTag的表达
将实施例1中构建好含有质粒(pET32a-L6KD-PT-MtuΔI-CM(WT) -LCB3-SpyTag、pET32a-L6KD-PT-MtuΔI-CM m1-LCB3-SpyTag、 pET32a-L6KD-PT-MtuΔI-CM m2-LCB3-SpyTag、pET32a-L6KD-PT-MtuΔI-CM m3-LCB3-SpyTag、pET32a-LCB3-SpyTag)的BL21(DE3)菌株接种到含100μg/mL羧苄青霉素的LB液体培养基中,并在37℃摇床中培养至对数期(OD600=0.4~0.6),加入终浓度0.2mM IPTG,培养条件为:18℃、2rpm,24小时后收获细胞,并测量菌浓度OD600(以下将1mL的OD600为1的细胞量称为1OD)。
将含有内含肽的菌体用裂解缓冲液B1(2.4g的Tris、29.22g的NaCl、0.37g 的Na2·EDTA·2H2O溶解于800mL水中,调pH至8.5,加水定容至1L)重悬至20 OD/mL,进行超声破碎(破碎条件为:功率200W,超声时间3sec,间隔时间3sec,超声次数99次)。在4℃,12000rpm的条件下离心20min,分别收集上清和沉淀部分。将沉淀用裂解缓冲液洗涤2次后,使用切割缓冲液(PBS补加40mM Bis-Tris, 2mM EDTA,调pH至6.2)充分重悬,置于25℃切割24h。之后将悬浊液离心分离,得到的上清和沉淀与切割前的沉淀一起进行SDS-PAGE检测(沉淀部分用与上一重悬步骤相同的体积的裂解缓冲液重悬)。
将直接表达LCB3-SpyTag融合蛋白的菌体用缓冲液Binding Buffer(20mM sodiumphosphate,0.5M NaCl,30mM imidazole,调pH至7.4)重悬至20OD/mL,进行超声破碎(破碎条件为:功率200W,超声时间3sec,间隔时间3sec,超声次数99次)。在4℃,12000rpm的条件下离心20min,分别收集上清和沉淀部分。得到的上清和与沉淀一起进行SDS-PAGE检测(沉淀部分用与上一重悬步骤相同的体积的缓冲液重悬)。
结果如图2A、图2B、图2C、图2D、图2E所示。图2A、图2B、图2C、图2D、图2E中显示含四种MtuΔI-CM突变株LCB3-SpyTag融合蛋白表达载体和 LCB3-SpyTag融合蛋白表达载体的纯度和产量。图2A是基于cSAT法采用MtuΔI-CM(WT)、MtuΔI-CM m1、MtuΔI-CM m2内含肽作为切割标签时的 LCB3-SpyTag融合蛋白表达纯化产物的SDS-PAGE结果;图2B是基于cSAT法采用MtuΔI-CM(WT)和MtuΔI-CM m1内含肽作为切割标签时的LCB3-SpyTag融合蛋白的表达纯化过程的SDS-PAGE结果;图2C是基于cSAT法采用MtuΔI-CM m2 内含肽作为切割标签时的LCB3-SpyTag融合蛋白的表达纯化过程的SDS-PAGE结果;图2D是基于cSAT法采用MtuΔI-CM m3内含肽作为切割标签时的LCB3-SpyTag 融合蛋白的表达纯化过程的SDS-PAGE结果;图2E是基于cSAT法采用MtuΔI-CM m3内含肽作为切割标签时的LCB3-SpyTag融合蛋白表达纯化产物和LCB3-SpyTag 直接表达的SDS-PAGE结果;WT泳道指示MtuΔI-CM(WT)切割上清产物,M1泳道是MtuΔI-CM m1的切割上清产物,M2泳道是MtuΔI-CM m2的切割上清产物, M3泳道是MtuΔI-CM m3的切割上清产物,S泳道指示直接表达上清产物。目的产物用黑框标示。ES:细胞裂解物上清;EP:细胞裂解物沉淀,可检测到清晰的融合蛋白表达成的聚集体;CP:表示的切割条件为25℃切割24h时,切割后分离的沉淀;CS:切割后分离的上清,图2B-图2D泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品,上样量依次为4μg、2μg、1μg、0.5μg、0.25μg。图2A和图2E泳道1-5为含有抑肽酶的蛋白定量标准品,上样量依次为4μg、2μg、1μg、0.5μg、 0.25μg。依照蛋白定量标准品,应用Bio-Rad公司的Quantity ONE凝胶定量分析软件对目的条带进行光密度分析,可计算得出融合蛋白形成的聚集体产量、在内含肽介导的自切割之后释放到上清中的LCB3-SpyTag融合蛋白的产量、MtuΔI-CM不同突变株的切割效率、LCB3-SpyTag融合蛋白在上清中的纯度,结果如表1所示。
表1 LCB3-SpyTag融合蛋白在25℃切割24h的条件下的表达与纯化情况
a蛋白聚集体产量,b内含肽介导的自切割后的LCB3-SpyTag产量,c内含肽介导的自切割效率=100%×(切割前聚集体表达量-切割后聚集体剩余量/切割前聚集体产量,d纯度=100%×LCB3-SpyTag的灰度/(LCB3-SpyTag的灰度+杂蛋白的灰度)。 /指在SDS-PAGE图上无法定量。
采用不同MtuΔI-CM突变株(MtuΔI-CM(WT)/MtuΔI-CM m1/MtuΔI-CM m2 /MtuΔI-CM m3)的LCB3-SpyTag融合蛋白均以沉淀形式存在,聚集体的表达量为 62~350mg/L,4种融合蛋白经内含肽MtuΔI-CM自切割,LCB3-SpyTag融合蛋白同MtuΔI-CM(WT)/MtuΔI-CM m1/MtuΔI-CM m2/MtuΔI-CM m3-L6KD分离,切割效率是8~81%。其中以MtuΔI-CM(WT)产量较高,且纯度较好,通过初步的cSAT 纯化,LCB3-SpyTag融合蛋白的产量为50mg/L,纯度达到65%。将使用Mtu ΔI-CM(WT)内含肽用于后续的实验。同时,直接表达LCB3-SpyTag融合蛋白的产量为65mg/L,也可以作为备选方法用于表达LCB3-SpyTag融合蛋白,组装多聚抗新冠病毒肽。
实施例3:构建N端与HisTag融合的抗新冠病毒肽LCB3单体(简称 LCB3-HisTag融合蛋白)的表达载体和融合多个SpyCatcher的ELP骨架蛋白表达载体
构建N端与HisTag融合的抗新冠病毒肽LCB3单体(简称LCB3-HisTag融合蛋白)的pET32a-LCB3-HisTag质粒以上述构建的pET30a-L6KD-PT-Mtu ΔI-CM(WT)-LCB3-SpyTag质粒为模板,通过Gibson assembly的方法构建,图3A为LCB3-HisTag融合蛋白表达载体的结构示意图。其中LCB3-HisTag融合蛋白的氨基酸如SEQ ID NO:27。以pET32a-L6KD-PT-MtuΔI-CM(WT)-LCB3-SpyTag质粒为模板,由于质粒载体自带一个Histag,利用引物(OY-21-002-F/OY-21-001-R,如SEQ ID NO:21/SEQ ID NO:22所示)通过PCR反应扩增LCB3-HisTag片段,利用引物(OY-21-001-F/OY-20-014-R,如SEQ ID NO:23/SEQ ID NO:24所示)通过PCR反应扩增AmpR-Ori片段,利用引物(OY-21-002-F/OY-20-014-R,如SEQ ID NO:21/ SEQ ID NO:23所示)通过重叠PCR反应扩增LCB3-HisTag-AmpR-Ori片段,利用引物(OY-20-014-F/OY-21-002-R,如SEQ ID NO:35/SEQ ID NO:26所示)通过PCR 反应扩增Lac I片段,以LCB3-HisTag-AmpR-Ori/Lac I片段进行Gibson assembly。将获得的连接产物化转入大肠杆菌DH5α,通过菌落PCR鉴定和测序筛选出正确的阳性克隆后,提取该质粒转化大肠杆菌表达菌株BL21(DE3)进行表达以及后续实验。
首先从文献(Zhongguang Yan等,2019)中获得融合两个SpyCatcher的ELP 蛋白的氨基酸序列,为了降低SpyCatcher的免疫原性,使用了文献(Long Li等, 2014)报道的N末端截短的SpyCatcher(ΔN)氨基酸序列进行改造,在N末端添加HisTag用于纯化,得到了SpyCatcher(ΔN)二聚蛋白(简称SpyC(ΔN)二聚蛋白)和 SpyCatcher(ΔN)三聚蛋白(简称SpyC(ΔN)三聚蛋白)氨基酸序列。将上述两种氨基酸序列进行大肠杆菌宿主密码子优化,优化后的SpyC(ΔN)二聚蛋白和SpyC(ΔN)三聚蛋白基因序列由上海生工合成,使用PET30a载体进行表达。
图3B和图3C是融合多个SpyCatcher的ELP骨架蛋白表达载体示意图,将上海生工合成的PET30a-HisTag-SpyCatcher(ΔN)-ELP-SpyCatcher(ΔN)和 PET30a-HisTag-SpyCatcher(ΔN)-ELP-SpyCatcher(ΔN)-ELP-SpyCatcher(ΔN)转入大肠杆菌BL21(DE3)转化进行表达以及后续实验。
实施例4:LCB3-HisTag融合蛋白、融合多个SpyCatcher的ELP骨架蛋白的表达
将实施例3构建好含有pET-LCB3-Histag的BL21(DE3)菌株接种到含100μg/mL 羧苄青霉素的LB液体培养基中,并在37℃摇床中培养至对数期(OD600=0.4~0.6),加入终浓度0.2mM IPTG,培养条件为:18℃、220rpm,24小时后收获细胞,并测量菌浓度OD600(以下将1mL的OD600为1的细胞量称为1OD)。
将实施例3构建好含有融合多个SpyCatcher的ELP骨架蛋白的BL21(DE3)菌株接种到含50μg/mL卡那霉素的LB液体培养基中,并在37℃摇床中培养至对数期(OD600=0.4~0.6),加入终浓度0.2mM IPTG,培养条件为:30℃、220rpm,6小时后收获细胞,并测量菌浓度OD600(以下将1mL的OD600为1的细胞量称为1OD)。
将LCB3-HisTag融合蛋白、融合多个SpyCatcher的ELP蛋白菌体用缓冲液 BindingBuffer(20mM sodium phosphate,0.5M NaCl,30mM imidazole,调pH 至7.4)重悬至20OD/mL,进行超声破碎(破碎条件为:功率200W,超声时间3sec,间隔时间3sec,超声次数99次)。在4℃,12000rpm的条件下离心20min,分别收集上清和沉淀部分。得到的上清和与沉淀一起进行SDS-PAGE检测(沉淀部分用与上一重悬步骤相同的体积的缓冲液重悬)。
SDS-PAGE检测结果如图4A和图4B所示,图中显示LCB3-HisTag融合蛋白、融合多个SpyCatcher的ELP蛋白表达载体的产量,可检测到清晰的融合蛋白表达,目的蛋白用黑框标示;P:破碎细胞的沉淀;S:破碎细胞的上清,图4A可检测到清晰的SpyC(ΔN)二聚蛋白条带,图4B可检测到清晰的SpyC(ΔN)三聚蛋白、LCB3-HisTag融合蛋白条带。图4A泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品,上样量依次为8μg、4μg、2μg、1μg、0.5μg。图4B泳道1-6为含有抑肽酶的蛋白定量标准品,上样量依次为8μg、4μg、2μg、1μg、0.5μg和0.25ug。
实施例5:多聚抗病毒肽LCB3的组装和纯化、LCB3-HisTag融合蛋白的纯化
多聚抗病毒肽LCB3的组装如图5A所示,将实施例2表达纯化的LCB3-SpyTag 融合蛋白和实施例3表达的将融合SpyCatcher的ELP蛋白裂解液在SDS-PAGE定量检测后,在LCB3-SpyTag融合蛋白过量的条件下,以SpyCatcher:SpyTag的摩尔比接近1:1的数量关系进行投料,在25℃,80rpm的条件下结合一夜。
结合产物用10K的超滤离心管(PALL Microsep)在4℃,4000rpm的条件多次离心,把溶液缓冲液交换为Binding Buffer,通过固相金属离子亲和层析(Immobilized metal-ion affinity chromatography,IMAC)对带有组氨酸标签的多聚抗新冠病毒肽进行制备性纯化,使用的是HisTrap FF层析柱,预装填料是Ni Sepharose 6Fast Flow。
将上述结合产物和实施例4的LCB3-HisTag融合蛋白裂解产物上样后,用 ElutionBuffer缓冲液(20mM sodium phosphate,0.5M NaCl,500mM imidazole,调pH至7.4)洗脱目的蛋白。条件为90%Binding buffer,10%Elution buffer可以洗脱LCB3-HisTag融合蛋白,条件为75%Binding buffer,15%Elution buffer可以洗脱二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽。
随后进行尺寸排阻色谱(Size-exclusion chromatography,SEC),使用分子筛柱(HiLoadTM 16/60SuperdexTM 75pg)对LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽进行精细纯化,用缓冲液(20mM Na2HPO4, 20mM NaH2PO4,pH 7.2)洗脱。通过两轮纯化的样品最终的SDS-PAGE检测如图5B所示,LH:两轮纯化后的LCB3-HisTag融合蛋白;L2:二聚LCB3抗新冠病毒肽;L3:三聚LCB3抗新冠病毒肽;泳道1-5为含有抑肽酶的蛋白定量标准品,上样量依次为8μg、4μg、2μg、1μg、0.5μg。
LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的分子量(Mw)和等电点(pI)、IMAC纯化后的纯度、回收率和SEC纯化后的纯度、回收率见表2。
表2 LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的纯化情况
实施例6:LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的生物膜层干涉实验(BLI)
以实施例5中由分子筛纯化得到的LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽样品为例,进行生物膜层干涉实验(BLI)。使用的是RED96e(ForteBio)分子间相互作用检测系统,将第2代氨基反应(AR2G)生物传感器浸入去离子水中进行平衡后,使用EDC/s-NHS激活,再浸入Loading Buffer(10mM acetate,0.02%Tween20,调pH至6)中,溶液中新冠病毒刺突蛋白 RBD(GenScript)结合到生物传感器表面,然后用1M的ethanolamine(调pH至 8.5)封闭传感器的结合位点便可进行检测。将固化完已知浓度抗原的传感器浸入 Running Buffer(10mM PBS,0.02%Tween20,0.1%BSA)使基线平稳,后将生物传感器浸入含有待测样品(LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽样品)的不同浓度溶液(6.25nM、12.5nM、25nM、50nM、 100nM)中,最后将已结合待测抗体的传感器浸入Running Buffer中进行解离,通过对实验过程中生物传感器生物膜层厚度的实时监控,可以得到待测样品与新冠病毒刺突蛋白蛋白RBD的动力学常数Kd、kon和koff。
LCB3-HisTag融合蛋白的BLI检测结果如图6A所示,二聚LCB3抗新冠病毒肽的BLI检测结果如图6B所示,三聚LCB3抗新冠病毒肽的BLI检测结果如图6C所示。LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽对新冠病毒刺突蛋白蛋白RBD的动力学常数Kd、kon和koff如表3所示。
表3.LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的动力学常数Kd、kon和koff
实施例7:LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的假病毒中和实验
以实施例5中由分子筛纯化得到的LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽样品为例,对含新冠病毒刺突蛋白假病毒和含新冠病毒刺突蛋白delta变体假病毒进行中和实验。
将LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽、三聚LCB3抗新冠病毒肽和对照品ACE2-Fc(Genscript)按照梯度稀释(100nM、25nM、6.25nM、1.5625nM、 0.390625nM、0.09765625nM、0.024414063nM、0.006103516nM)取假病毒与样品或对照品在96孔细胞培养中混合。在室温下孵育混合溶液1h。孵育结束后,将过表达ACE2细胞系加入已完成中和反应的孔,使细胞数达到20,000个/孔。在37℃, 5%CO2培养箱中孵育24h。24小时后,取出96孔板并加入50μl新鲜的培养基。在37℃,5%CO2培养箱中继续孵育24h。48h后,从培养箱中取出检测96孔板检测中和反应效果。去除培养基后,立即加入50μl Bio-GloTM检测试剂到对应孔中,室温孵育3-5min。用PHERAStar或EnVision酶标仪机进行读板检测。
LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽、三聚LCB3抗新冠病毒肽的含新冠病毒刺突蛋白假病毒中和实验检测结果如图7A所示,含新冠病毒刺突蛋白delta变体假病毒中和实验检测结果如图7B所示。
LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒和含新冠病毒刺突蛋白delta变体假病毒中和实验检测的半数抑制浓度(IC50)如表4所示。
表4.LCB3-HisTag融合蛋白、二聚LCB3抗新冠病毒肽和三聚LCB3抗新冠病毒肽的半数抑制浓度(IC50)
以上实施例仅为本发明较优的实施方式,仅用于解释本发明,而非限制本发明,本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
序列表
<110> 华南理工大学
<120> 一种多聚肽及其制备方法与应用
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 64
<212> PRT
<213> 人工序列(Artificial Sequence)
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Asn Asp Asp Glu Leu His Met Leu Met Thr Asp Leu Val Tyr Glu Ala
1 5 10 15
Leu His Phe Ala Lys Asp Glu Glu Ile Lys Lys Arg Val Phe Gln Leu
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Phe Glu Leu Ala Asp Lys Ala Tyr Lys Asn Asn Asp Arg Gln Lys Leu
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Glu Lys Val Val Glu Glu Leu Lys Glu Leu Leu Glu Arg Leu Leu Ser
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<210> 2
<211> 78
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
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Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
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Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly
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Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
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Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
65 70 75
<210> 3
<211> 91
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 3
Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu
1 5 10 15
Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile
20 25 30
Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro
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Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val
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Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val
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Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile
85 90
<210> 4
<211> 118
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
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Val Thr Thr Leu Ser Gly Leu Ser Gly Glu Gln Gly Pro Ser Gly Asp
1 5 10 15
Met Thr Thr Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg
20 25 30
Asp Glu Asp Gly Arg Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp
35 40 45
Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly His Val Lys
50 55 60
Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala
65 70 75 80
Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu
85 90 95
Gln Gly Gln Val Thr Val Asn Gly Glu Ala Thr Lys Gly Asp Ala His
100 105 110
Thr Gly Ser Ser Gly Ser
115
<210> 5
<211> 118
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 5
Val Thr Thr Leu Ser Gly Leu Ser Gly Glu Gln Gly Pro Ser Gly Asp
1 5 10 15
Met Thr Thr Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg
20 25 30
Asp Glu Asp Gly Arg Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp
35 40 45
Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly His Val Lys
50 55 60
Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala
65 70 75 80
Pro Asp Gly Tyr Glu Val Ala Thr Pro Ile Glu Phe Thr Val Asn Glu
85 90 95
Asp Gly Gln Val Thr Val Asp Gly Glu Ala Thr Glu Gly Asp Ala His
100 105 110
Thr Gly Ser Ser Gly Ser
115
<210> 6
<211> 13
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 6
Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210> 7
<211> 14
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 7
Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys
1 5 10
<210> 8
<211> 16
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 8
Arg Gly Val Pro His Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys
1 5 10 15
<210> 9
<211> 17
<212> PRT
<213> 化脓性链球菌(Streptococcus pyogenes)
<400> 9
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
1 5 10 15
Pro
<210> 10
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Leu Leu Leu Leu Leu Leu Lys Asp
1 5
<210> 11
<211> 168
<212> PRT
<213> 结核分枝杆菌(M.tuberculosis)
<400> 11
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Leu Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
115 120 125
Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
130 135 140
Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Thr Leu Val
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Ala Glu Gly Val Val Val His Asn
165
<210> 12
<211> 168
<212> PRT
<213> 结核分枝杆菌(M.tuberculosis)
<400> 12
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Leu Trp Ala Thr Pro Asp Tyr Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
115 120 125
Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
130 135 140
Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Val Leu Val
145 150 155 160
Ala Glu Gly Val Val Val His Asn
165
<210> 13
<211> 168
<212> PRT
<213> 结核分枝杆菌(M.tuberculosis)
<400> 13
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
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Ala Ile Leu Trp Ala Thr Pro Asp Val Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
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Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
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Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Ser Leu Val
145 150 155 160
Ala Glu Gly Val Val Val His Asn
165
<210> 14
<211> 168
<212> PRT
<213> 结核分枝杆菌(M.tuberculosis)
<400> 14
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Leu Trp Ala Thr Pro Asp Val Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
115 120 125
Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
130 135 140
Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Cys Leu Val
145 150 155 160
Ala Glu Gly Val Val Val His Asn
165
<210> 15
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggcgttgtgg ttcataacaa cgatgacgaa ctgcacatgc t 41
<210> 16
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atcaaccatg acaatatggg cgctcagcag acgttccag 39
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcccatattg tcatggttga tgcat 25
<210> 18
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
attaccccca tgaacagaaa tcccc 25
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gggggatttc tgttcatggg ggtaa 25
<210> 20
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ttatgaacca caacgccttc cgca 24
<210> 21
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aagaaggaga tatacatatg aacgatgacg aactgcacat gctgatgac 49
<210> 22
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agtggtggtg gtggtggtgc tcgaggctca gcagacgttc cagcagtt 48
<210> 23
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tcgagcacca ccaccaccac cact 24
<210> 24
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ccgggcatgt tcatcatcag taacccgta 29
<210> 25
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tcgagcacca ccaccaccac cact 24
<210> 26
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccgggcatgt tcatcatcag taacccgta 29
<210> 27
<211> 73
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Asn Asp Asp Glu Leu His Met Leu Met Thr Asp Leu Val Tyr Glu
1 5 10 15
Ala Leu His Phe Ala Lys Asp Glu Glu Ile Lys Lys Arg Val Phe Gln
20 25 30
Leu Phe Glu Leu Ala Asp Lys Ala Tyr Lys Asn Asn Asp Arg Gln Lys
35 40 45
Leu Glu Lys Val Val Glu Glu Leu Lys Glu Leu Leu Glu Arg Leu Leu
50 55 60
Ser Leu Glu His His His His His His
65 70
Claims (10)
1.一种多聚肽的制备方法,其特征在于,包括如下步骤:
(a)构建及表达融合SpyTag肽的目标多肽;
(b)构建及表达融合SpyCatcher肽和ELP肽的骨架蛋白;将ELP肽和SpyCatcher肽的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞表达,得到工程菌,超声裂解,进行金属亲和层析和尺寸排阻色谱纯化分离;
(c)混合目标多肽和骨架蛋白,自连接形成多聚肽;
(d)纯化获得高纯度多聚肽;
所述的目标多肽是抗体、纳米抗体、蛋白、抗菌肽或抗病毒肽。
2.根据权利要求1所述多聚肽的制备方法,其特征在于,所述抗病毒肽是抗新型冠状病毒肽,所述抗新型冠状病毒肽为LCB3多肽(SEQ ID NO:1)。
3.根据权利要求1所述多聚肽的制备方法,其特征在于,所述骨架蛋白的SpyCatcher肽和ELP肽以交替重复顺序融合,其重复顺序特征为:
(a)SpyCatcher肽的重复次数为n次,ELP肽的重复次数为n-1至n+1次;其中n大于或等于2;
(b)骨架蛋白的重复顺序为A-B-A、A-B-A-B-A或B-A-B-A-B、B-A-B-A-B-A-B,A代表SpyCatcher肽,B代表ELP肽。
4.根据权利要求1所述多聚肽的制备方法,其特征在于,所述ELP肽的特征满足如下条件:
(a)Val-Pro-Gly-Xaa-Gly的重复序列;
(b)Val-Pro-Gly-Xaa-Gly重复序列的重复数为10~30次,优选12~18次;
(c)其中Xaa为Val和Glu;
(d)其中Xaa氨基酸Val和Glu的比例3:1~5:1;
(e)ELP肽为SEQ ID NO:2所示的氨基酸序列。
5.根据权利要求1或3所述多聚肽的制备方法,其特征在于,SpyCatcher肽与所述的SpyTag肽形成异肽链,所述SpyCatcher肽包含选自SEQ ID NO:3,4,5任一所示的氨基酸序列,所述SpyTag肽包含选自SEQ ID NO:6,7,8任一所示的氨基酸序列。
6.根据权利要求1所述多聚肽的制备方法,其特征在于,所述融合SpyTag肽的目标多肽的制备方法,包括如下步骤:
(a)将目标多肽、SpyTag肽和纯化标签的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌;培养所述工程菌表达所述融合蛋白,然后裂解工程菌,捕获得到所述融合蛋白;
所述的纯化标签由聚集肽和切割标签组成;所述聚集肽的基因序列与切割标签的基因序列之间通过接头连接,所述接头为PT型接头;所述PT型接头为如SEQ ID NO:9所示氨基酸序列;
所述聚集肽为两亲性自组装短肽,所述两亲性自组装短肽为类表面活性剂短肽;所述类表面活性剂短肽为如SEQ ID NO:10所示氨基酸序列;
所述切割标签为化学切割位点、酶法切割位点或自切割位点,所述自切割位点为内含肽,所述内含肽为MtuΔI-CM内含肽或其突变体为如SEQ ID NO:11,12,13,14所示氨基酸序列;
所述捕获得到融合蛋白,包括将工程菌裂解后离心取沉淀,得到融合蛋白的聚集体;将所述融合蛋白的聚集体进行切割;
所述切割,包括:将所述融合蛋白的聚集体分散在缓冲液中,进行切割处理,离心取上清液,得到所述融合蛋白的溶液;所述缓冲液的pH值为5.5~6.8,所述切割处理的温度为4~37℃,切割处理的时间为3~48h。
7.根据权利要求1所述多聚肽的制备方法,其特征在于,所述骨架蛋白的制备方法包括如下步骤:
(a)将SpyCatcher肽,ELP肽和纯化标签的基因序列依次连接,形成融合蛋白的基因,将所述融合蛋白的基因导入宿主细胞,得到工程菌;所述纯化标签为亲和纯化标签,所述亲和纯化标签为HisTag标签;
(b)所述工程菌表达所述融合蛋白,然后裂解工程菌,得到所述融合蛋白的溶液。
8.根据权利要求1所述多聚肽的制备方法,其特征在于,所述多聚肽的制备方法,包括如下步骤:
(a)将融合SpyTag肽的目标多肽和骨架蛋白,按各自融合蛋白所具有的SpyTag和SpyCatcher的摩尔比的1~1.5倍进行混合反应,得到多聚肽溶液;所述混合反应的温度为4~37℃,混合反应的时间为3~24h;
(b)将多聚肽溶液,通过金属亲和层析和尺寸排阻色谱进行纯化,得到高纯度的多聚肽。
9.权利要求1~8任一项所述方法制备得到多聚肽,其特征在于,所述多聚肽为多聚抗新冠病毒肽,所述多聚抗新冠病毒肽为二聚LCB3抗新冠病毒肽或三聚LCB3抗新冠病毒肽;
所述二聚LCB3抗新冠病毒肽对新冠病毒刺突蛋白RBD的解离常数Kd小于1pM,所述三聚LCB3抗新冠病毒肽对新冠病毒刺突蛋白RBD的解离常数Kd小于1pM。
所述二聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒的半数抑制浓度IC50为0.57nM,所述三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白假病毒的半数抑制浓度IC50为0.20nM。
所述二聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白delta变体假病毒的半数抑制浓度IC50为0.53nM,所述三聚LCB3抗新冠病毒肽对含新冠病毒刺突蛋白delta变体假病毒的半数抑制浓度IC50为0.32nM。
10.权利要求9所述多聚肽应用于新冠病毒感染的防控与治疗。
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