CN114456263A - 一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用 - Google Patents
一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用 Download PDFInfo
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Abstract
本发明适用于基因工程技术领域,提供了一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用,纳米抗体的核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示,纳米抗体的氨基酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。本发明的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体可以特异性结合人、猪和兔HEV 239蛋白,利用原核表达系统表达并纯化纳米抗体,与猪HEV 239蛋白或猪HEV分别孵育后接种HpG2细胞,从而发挥抗病毒功能。
Description
技术领域
本发明属于基因工程技术领域,尤其涉及一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用。
背景技术
戊型肝炎(Hepatitis E)是由戊型肝炎病毒(Hepatitis E virus,HEV)感染引起的一种人兽共患性疾病,全球每年约有 2000 万人感染,其中 330 万人出现临床症状,4.4万人死亡(WHO: WHO fast sheet: Hepatitis E virus. 2019.)。HEV 感染的流行病学调查发现,近几年人群中 HEV 感染已转变为以食源性传播为主,猪是其主要的自然储存宿主。因此从源头控制动物源性 HEV 的传播流行显得尤为重要。然而目前对猪HEV的防控,仍以清理传染源、减少应激等预防措施为主,尚无特效疫苗和治疗药物。HEV 为单股正链不分节段 RNA 病毒(7.2kb),其基因组共编码 3 个开放阅读框(Open Reading frame,ORF),其中ORF2 编码包含着显性表位的结构蛋白,在诱导体液免疫保护中发挥着重要作用(WangX.Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Proteinof Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and HumanHEVs. J Virol. 2015; 89: 54)。因此,目前针对 HEV 疫苗及其抗体研发都是基于重组表达全长或截短的 HEV ORF2 衣壳蛋白(HEV 239 蛋白:位于 HEV ORF2 的氨基酸区域 aa368-606,通过大肠杆菌原核表达后在一定盐离子浓度条件下可自我组装形成病毒样粒子(VLP),并模拟病毒表面的抗原结构,因此被研发成为目前市场上针对 HEV 的疫苗益可宁。)(Shrestha MP. Safety and efficacy of a recombinant hepatitis E vaccine. NEngl J Med. 2007; 356:895-903.)。
骆驼科动物体内存在一种天然缺失轻链和重链第一个恒定区的特殊 IgG,被称作重链抗体(Heavy chain-only antibodies,HcAbs),与传统的抗原结合片段不同,其抗原结合片段由一个单独的结构域构成,即重链抗体可变区(Variable domains of Camellidaeheavy chain-only antibodies,VHH)(Hamers CC.Naturally occurring antibodiesdevoid of light chains. Nature. 1993; 363(6428):446-8.)。VHH 分子量仅有15 kDa左右,呈橄榄球状,直径约 2.5 nm,高约 4 nm,是已知最小的具有完整抗原结合功能的抗体片段,因此被称为纳米抗体(nanobody)(Muyldermans S.Camelid immunoglobulins andnanobody technology. Vet Immunol Immunopathol. 2009; 128(1-3):178-83.)。纳米抗体的特殊结构使其具备一般抗体所不具有的特性,如分子量小、高水溶性、高耐性、结构稳定、较强的组织穿透力、易于基因工程改造、生产成本低以及可以识别特殊抗原表位等优点,使得该抗体作为第三代抗体在基础研究、药物开发等领域有广阔的应用前景。目前对纳米抗体的应用主要依靠其结构和功能特异性,已被广泛应用于免疫学检测、肿瘤的诊断及治疗和抗病毒研究等领域(Muyldermans S. Nanobodies: natural single-domainantibodies. Annu Rev Biochem. 2013; 82:775-97. Hassanzadeh GG, Devoogdt N, DePauw P. Nanobodies and their potential applications. Nanomedicine (Lond).2013; 8(6):1013-26. Vanlandschoot P. Nanobodies(R): new ammunition to battleviruses. Antiviral Res. 2011; 92(3):389-407.)。由 Ablynx 公司研发的针对呼吸道合胞病毒(RSV)的纳米抗体ALX-0171,可以结合病毒表面的融合蛋白从而抑制病毒的复制,是第一个处于临床实验阶段的抗病毒纳米抗体药物,与传统单克隆抗体相比 ALX-0171 具有更强和更广谱的中和活性(Detalle L. Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of RespiratorySyncytial Virus Infection. Antimicrob Agents Ch. 2015; 60(1):6-13.)。Tarr 等分离到 1 株针对甲型肝炎病毒 E2 蛋白的纳米抗体(D03),可以干扰病毒 E2 蛋白与 CD81的相互作用,与一般的中和抗体不同,D03 不仅可以中和病毒感染,还可以抑制病毒在细胞间的传播(Tarr AW. An alpaca nanobody inhibits hepatitis C virus entry andcell-to-cell transmission. Hepatology. 2013; 58(3):932-9.)。Jittavisutthikul等制备了可以穿透细胞膜的 HCV 丝氨酸蛋白酶的人源化纳米抗体,这些纳米抗体进入细胞后可以抑制 HCV 的复制,并且可以减弱 HCV 对感染细胞的免疫抑制作用(Jittavisutthikul S. Humanized-VHH Transbodies that Inhibit HCV Protease andReplication. Viruses-Basel. 2015; 7(4):2030-56.)。
近年来,不断有抗 HEV 的中和抗体被研究发现,但仍未有相关的抗体药物投入生产使用。传统抗体结构复杂、分子量较大、研发和生产成本高,这些缺陷限制其在药物生产等方面的发展应用。
发明内容
本发明实施例的目的在于提供一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用,旨在解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体,纳米抗体的核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示,纳米抗体的氨基酸序列分别如SEQ ID NO:3和SEQ IDNO:4所示。
进一步的,所述纳米抗体通过表达载体表达。
进一步的,所述纳米抗体与人、猪和兔HEV 239蛋白特异性结合。
进一步的,所述纳米抗体能够抑制猪HEV 239蛋白或猪HEV吸附HepG2细胞。
进一步的,一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体在抗病毒感染中的应用。
与现有技术相比,本发明的有益效果是:
该特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用,本发明的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体可以特异性结合人、猪和兔HEV 239蛋白,利用原核表达系统表达并纯化纳米抗体,与猪HEV 239蛋白或猪HEV分别孵育后接种HpG2细胞,从而发挥抗病毒功能。
附图说明
图1为SDS和WB实验鉴定纳米抗体表达的结果。
图2为ELISA实验鉴定纳米抗体结合力及特异性的结果。其中Kernow-239(基因3型)和Sar-55-239(基因1型)是人HEV蛋白,rHEV-239 (基因3型)是兔HEV蛋白,sp239(基因4型)是猪HEV蛋白;1B5单抗与上述蛋白反应作为阳性对照;nb anti-PCV2作为阴性对照。
图3为WB实验鉴定纳米抗体抑制猪HEV 239蛋白吸附HepG2细胞的结果。
图4为IFA实验鉴定纳米抗体抑制猪HEV 239蛋白吸附HepG2细胞的结果。
图5为qRT-PCR实验鉴定纳米抗体抑制猪HEV吸附HepG2细胞的结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
本发明首先将猪HEV 239重组蛋白免疫一只阿拉善双峰驼,经过5次免疫后分离外周血淋巴细胞,并构建特异的单域重链抗体文库,利用噬菌体展示技术筛选并获得了针对猪HEV 239蛋白特异性纳米抗体。构建携带纳米抗体基因的原核表达载体,表达纯化后通过ELISA检测这些纳米抗体与人、猪和兔HEV 239蛋白结合。将这些原核表达的纳米抗体分别与猪HEV 239蛋白或猪HEV孵育后接种HepG2细胞,通过IFA、WB和qRT-PCR检测方法揭示这些纳米抗体可以有效抑制蛋白或病毒吸附宿主细胞。
实施例1
抗猪HEV衣壳蛋白纳米抗体的筛选:
将表达纯化后的猪HEV 239蛋白免疫双峰驼,检测抗体效价达标后采集外周血分离淋巴细胞并提取细胞基因组RNA,采用巢式RT-PCR扩增编码VHH的基因片段,连入pCANTAB5E构建重组噬菌体载体并电转入TG1感受态细胞,构建双峰驼重链抗体可变区文库;利用噬菌体展示技术淘选针对猪HEV 239蛋白的特异性纳米抗体。最终得到两株针对猪HEV 239蛋白的特异性纳米抗体,命名为猪HEV-nb1和猪HEV-nb2。核苷酸序列分别如SEQ IDNO:1和SEQ ID NO:2所示,氨基酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。
实施例2
原核表达纳米抗体:
以pET-21b作为原核表达载体,构建好的纳米抗体重组质粒转化至BL21(DE3)表达感受态细胞,37℃,220 rpm诱导表达6-8 h。由于纳米抗体主要以包涵体形式表达,首先用8M尿素溶解沉菌并通过镍柱纯化,然后梯度透析至0 .01M PBS(PH7 .2)进行复性,浓缩后用0 .22 μm无菌滤膜过滤除菌,最后在-80℃保存待用。SDS和WB结果如图1显示,在15 kDa附近出现特异性目的条带,说明融合蛋白表达。
实施例3
ELISA检测纳米抗体与人、猪和兔HEV 239蛋白结合:
由于人、猪和兔HEV 239蛋白之间的同源性高达93%以上。将人、猪和兔HEV 239蛋白分别每孔400ng包板过夜,封闭洗板后分别加入纳米抗体,后加入鼠抗his抗体及羊抗鼠HRP标记的二抗显色鉴定。结果如图2显示,纳米抗体与人、猪和兔HEV 239蛋白均进行特异性反应。说明这些纳米抗体具有广谱性。
实施例4
具有抗猪HEV 239蛋白或猪HEV中和活性纳米抗体的验证:
HepG2细胞铺板后将猪HEV 239蛋白与纳米抗体混合液一起加入板内,4℃孵育1h后利用2×sample buffer刮取细胞,煮样,跑胶转膜后加入鼠抗猪HEV衣壳蛋白单抗3E8及羊抗鼠HRP标记的二抗显色。结果如图3显示,与纳米抗体混合后再与HepG2孵育的猪HEV239蛋白量明显少于阴性对照组的蛋白量。HepG2细胞铺板,纳米抗体与猪HEV 239蛋白提前孵育3h后加入板内,4℃静置1h,利用4%多聚甲醛固定细胞后用0.25% TrionX-100进行破膜,2.5% BSA封闭后,加入鼠抗HEV-ORF2蛋白单抗3E8孵育过夜后加入红色荧光二抗,最后利用含DAPI的商品化封片剂进行封片,于荧光显微镜下进行观察。结果如图4显示,与纳米抗体混合后再与HepG2孵育的猪HEV-239的荧光强度明显低于阴性对照组的荧光强度。将上述纳米抗体与猪HEV的病毒悬液37℃孵育30 min后,将混合物移至宿主细胞HepG2继续孵育30 min,然后将未结合的病毒用PBS洗去,再用qRT-PCR技术检测病毒RNA。结果如图5显示,与纳米抗体混合后再与HepG2孵育的猪HEV拷贝数明显低于阴性对照组的拷贝数。
本发明的工作原理是:
该特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体及应用,本发明的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体可以特异性结合人、猪和兔HEV 239蛋白,利用原核表达系统表达并纯化纳米抗体,与猪HEV 239蛋白或猪HEV分别孵育后接种HpG2细胞,从而发挥抗病毒功能。
以上仅是本发明的优选实施方式,应当指出,对于本领域的技术人员来说,在不脱离本发明构思的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些均不会影响本发明实施的效果和专利的实用性。
Claims (5)
1.一种特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体,其特征在于,纳米抗体的核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示,纳米抗体的氨基酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。
2.根据权利要求1所述的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体,其特征在于,所述纳米抗体通过表达载体表达。
3.根据权利要求2所述的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体,其特征在于,所述纳米抗体与人、猪和兔HEV 239蛋白特异性结合。
4.根据权利要求3所述的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体,其特征在于,所述纳米抗体能够抑制猪HEV 239蛋白或猪HEV吸附HepG2细胞。
5.一种根据权利要求1-4任一所述的特异性结合戊型肝炎病毒衣壳蛋白的纳米抗体在抗病毒感染中的应用。
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