CN107022007A - 基于马尔堡病毒包膜蛋白的抗原片段、截短体及应用 - Google Patents
基于马尔堡病毒包膜蛋白的抗原片段、截短体及应用 Download PDFInfo
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Abstract
本发明公开了一种基于马尔堡病毒包膜蛋白的抗原片段、截短体及应用。本发明提供的抗原片段GP2具有与GP蛋白相似的抗原潜力,免疫产生的中和抗体能够有效抑制病毒的感染。与完整的GP蛋白抗原相比,该抗原片段不具有GP蛋白的生物学功能,因此不能使病毒进入宿主细胞,该片段用于病毒载体疫苗的构建时具有更好的安全性,也更方便用于多价疫苗制备。
Description
技术领域
本发明涉及一种基于马尔堡病毒包膜蛋白的抗原片段、截短体及应用,属于生物技术领域。
背景技术
马尔堡病毒(Marburg virus,MARV)属于丝状病毒科,为单股不分节段负链RNA病毒,在电镜下呈细丝状,是烈性传染病马尔堡出血热的病原体。该病毒在1967年由德国科学家发现(Siegert R,Shu HL.Ger Med Mon,1968,13(1):1–2.),迄今已在非洲等地出现散在性暴发十余次,感染造成数百人死亡,死亡率大于80%,2005年在安哥拉暴发的马尔堡出血热死亡率高达90%(Jonathan S.Towner ML.Virol,2006,80(13):6497–6516.)。MARV感染后会导致严重的出血热,破坏人体的多种脏器和免疫系统。自然状态下该病毒主要通过直接接触患者的体液传播,病毒感染后,相关疾病症状很快出现,包括头痛、肌痛、发热、多器官功能衰竭等。目前,没有有效的疫苗及药物用于马尔堡病毒感染的预防及治疗。
马尔堡病毒基因组全长19.1Kb,基因组分别编码核衣壳蛋白(Nucleoprotein,NP)、病毒蛋白(viral protein)VP35、VP40、包膜糖蛋白(Glycoprotein,GP)、VP30、VP24以及聚合酶大蛋白L(Mire CE,Geisbert JB,PLoS ONE 9(4):e94355.)。病毒颗粒表面被有包膜,包膜上覆盖着由GP三聚体组成的长度为5-10nm的棘突。病毒颗粒的核心部位是基因组RNA和与其紧密连接的核衣壳蛋白组成核衣壳复合体,紧密连接能防止其被RNase降解,这些蛋白包括NP、VP30、VP35和L。GP蛋白属Ⅰ型跨膜蛋白,GP处在病毒包膜外侧,能介导病毒与目标细胞的粘附与融合。GP为病毒外部唯一蛋白,是主要抗原部分,是目前研究疫苗首选抗原。GP全长基因有2046个碱基,由一个ORF编码合成681aa,形成的GP前体被Furin酶切割(位点为435aa与436aa之间)形成GP1和GP2两个亚基,由二硫键连接形成一个GP单体,三个GP单体构成一个成熟的GP。GP1分子量大约为160KDa,存在大量抗原表位,其aa1-18为信号肽,aa33-188为受体结合区(Receptor binding domain,RBD)。受体结合区被证明是抗体直接作用的主要位点(Flyak AI,Ilinykh PA.Cell,2015,160(5):893–903.),是介导病毒黏附的关键区域,此区域糖基化位点较多。aa277-455是黏蛋白样区(mucin-like domain,MLD),此区域高度糖基化,覆盖在GP1和GP2之间,逃逸免疫作用,该区域存在增强抗体表位,能增强病毒的感染效率。病毒一旦进入细胞,胞内的组织蛋白酶识别GP,切割其上面糖帽和黏蛋白样区域,切割后的GP识别胞内NPC1受体,使原本被紧密束缚的GP2发生重排,形成六螺旋束,催化病毒与细胞膜融合(Takao Hashiguchi,Marnie L.Fusco.2015,Cell 160,904–912)。
近十年来,随着马尔堡病毒致病机制的阐明,目前,马尔堡病毒疫苗的研究已取得了显著进展,已研究的疫苗包括灭活疫苗、DNA疫苗、病毒颗粒样蛋白疫苗、rAD载体疫苗、rVSV载体疫苗等,抗原主要为包膜糖蛋白GP。近期抗体-马尔堡GP蛋白相互作用及结构测定结果表明,马尔堡GP的aa33-188受体结合区(Receptor binding domain,RBD)为抗体直接作用的主要位点(Hashiguchi et al.,2015,Cell 160,904–912)。由于嵌入完整GP蛋白的病毒载体疫苗具有一定的复制能力及恢复毒性的风险,安全问题是这类疫苗不可忽视和亟待克服的问题。
发明内容
本发明的第一个目的是提供如下M1)或M2)或M3)所示的蛋白质:
M1)马尔堡病毒的GP蛋白自N端起第436-648位氨基酸;
M2)马尔堡病毒的GP蛋白自N端起第402-562位氨基酸;
M3)马尔堡病毒的GP蛋白自N端起第19-648位氨基酸。
本发明的第二个目的是提供一种蛋白质或其截短体。
上述蛋白质或其截短体中,
所述截短体为如下(1)-(5)中任一所示:
(1)氨基酸序列是序列2第436-648位所示的蛋白质;
(2)氨基酸序列是序列2第402-562位所示的蛋白质;
(3)在序列2第436-648位或序列2第402-562位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
(4)将序列2第436-648位或序列2第402-562位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质;
(5)与序列2第436-648位或序列2第402-562位所示的氨基酸序列具有90%以上的同一性且具有相同功能的蛋白质;
所述蛋白质为如下(6)-(9)中任一所示:
(6)氨基酸序列是序列2第19-648位所示的蛋白质;
(7)在序列2第19-648位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
(8)将序列2第19-648位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质;
(9)与序列2第436-648位或序列2第402-562位所示的氨基酸序列具有90%以上的同一性且具有相同功能的蛋白质。
本发明的第三个目的是提供上述蛋白质或其截短体的编码基因。
上述编码基因中,上述M1)或M2)所述蛋白质或上述截短体的编码基因为如下1)-4)中任一所示:
1)序列1中自5’末端起第1306位至第1944位核苷酸所示的DNA分子;
2)序列1中自5’末端起第1204位至第1686位核苷酸所示的DNA分子;
3)在严格条件下与1)或2)限定的DNA分子杂交且编码M1)或M2)所述蛋白质或截短体的DNA分子;
4)与1)或2)或3)限定的DNA分子具有90%以上的同一性且编码M1)或M2)所述蛋白质或截短体的DNA分子;
上述蛋白质的编码基因为如下5)-7)任一所示:
5)序列1中自5’末端起第55-1944位核苷酸所示的DNA分子;
6)在严格条件下与5)限定的DNA分子杂交且编码上述蛋白质的DNA分子;
7)与5)或6)限定的DNA分子具有90%以上的同一性且编码上述蛋白质的DNA分子。
本发明的第四个目的是提供下述(a1)-(a4)中的任一种生物材料:
(a1)含有上述编码基因的表达盒;
(a2)含有上述编码基因的重组载体;
(a3)含有上述编码基因的重组菌;
(a4)含有上述编码基因的转基因细胞系。
本发明的第五个目的是提供上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料的新用途。
本发明提供了上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料在制备免疫原和/或抗原中的应用。
上述应用中,所述免疫原或抗原是针对马尔堡病毒或马尔堡病毒包膜蛋白GP的。
本发明还提供了上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料作为抗原在制备针对马尔堡病毒的抗体中的应用。
本发明还提供了上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料在制备预防和/或治疗马尔堡病毒引起的疾病的产品中的应用。
本发明还提供了上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料在预防和/或治疗马尔堡病毒引起的疾病中的应用。
本发明还提供了上述蛋白质或其截短体和/或上述编码基因和/或上述生物材料在如下a1)-a6)中任一种中的应用:
a1)抑制病毒感染;
a2)制备抑制病毒感染的产品;
a3)抑制病毒与宿主细胞结合;
a4)制备抑制病毒与宿主细胞结合的产品;
a5)抑制病毒入侵;
a6)制备抑制病毒入侵的产品。
本发明的第六个目的是提供一种产品。
本发明提供的产品的活性成分为上述蛋白质或其截短体;
所述产品的功能为如下b1)-b3)中任一种:
b1)抑制病毒感染;
b2)抑制病毒与宿主细胞结合;
b3)抑制病毒入侵;
所述产品为药物或疫苗。
上述应用或上述产品中,
所述病毒为马尔堡病毒。
本发明的第七个目的是提供一种多抗。
本发明提供的多抗是以上述蛋白质或其截短体为免疫原制备得到的。
本发明发现暴露于GP蛋白表面的后部区域(aa436-648)在GP蛋白功能的发挥中具有关键作用,且该区域含有丰富的GP蛋白抗原表位。本发明提供的抗原片段是来自马尔堡病毒包膜蛋白GP的一段序列(aa436-648)。本发明提供的抗原片段GP2具有与GP蛋白相似的抗原潜力,免疫产生的中和抗体能够有效抑制病毒的感染。与完整的GP蛋白抗原相比,该抗原片段不具有GP蛋白的生物学功能(不具有GP1亚基与GP2亚基的功能,在病毒进入过程中只发挥辅助功能),因此不能使病毒进入宿主细胞,该片段用于病毒载体疫苗的构建时具有更好的安全性,也更方便用于多价疫苗制备。
附图说明
图1为1%琼脂糖凝胶分析pVAX1-GP△,pVAX1-RBS,pVAX1-RBSP,pVAX1-FU,pVAX1-空载体的电泳图。
图2为重组蛋白GP1△的表达及纯化后SDS-PAGE电泳分析图。
图3为重组蛋白GP2△的表达及纯化后SDS-PAGE电泳分析图。
图4为重组蛋白GPM的表达及纯化后SDS-PAGE电泳分析图
图5为重组质粒第三次免疫小鼠血清的ELISA分析图。
图6为GP蛋白和GP1△、GP2△、GPM蛋白第三次免疫小鼠血清的ELISA分析图。
图7为Concanavalin A(ConA)或GP蛋白刺激后GP免疫组与GP2△免疫组的淋巴细胞刺激指数分析。图7A为ConA刺激后GP免疫组与GP2△免疫组的淋巴细胞刺激指数分析:图7B为GP蛋白刺激后GP免疫组与GP2△免疫组的淋巴细胞刺激指数分析。
图8为pVAX1-GP△,pVAX1-FU,GP,GP2△免疫组免疫血清对假病毒感染靶细胞的中和活性分析图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中所用的酶,除特殊说明外,均购自NEB公司。
下述实施例中所用的细胞培养基及血清,除特殊说明外,均购自Gibco公司。
下述实施例中所用的载体pVAX1购自Invitrogen公司。
下述实施例中所用的载体pUC57-MGP从金唯智公司购买,MGP基因的GenBank号为DQ447659.1。
下述实施例中所用的6周龄Balb/C雌性小鼠购自中国人民解放军军事医学科学院实验动物中心,许可证号:SCXK-(军)2012-0004。
下述实施例中所用的IL-2ELISA kit、IFN-γELISA kit、小鼠淋巴细胞分离液和Cell Counting Kit TetraZ均购自达科为生物技术公司,产品目录号分别为:DKW12-2020,DKW12-2000、DKW33-R0100和424501。
下述实施例中所用的Goat-antimouse IgG HRP-conjugated antibody、Goat-antimouse IgG1HRP-conjugated antibody和Goat-antimouse IgG2a HRP-conjugatedantibody均购自Abcam公司。
下述实施例中所用的Britelite plus Luminesence Reporter Gene AssaySystem购自PE公司,产品目录号为6066766。
下述实施例中所用的HEK293T细胞购自Cloneth。
下述实施例中所用的pNL4-3.luc.RE质粒在文献“L.Du et al.,Development ofa safe and convenient neutralization assay for rapid screening of influenzaHA-specific neutralizing monoclonal antibodies,Biochemical and BiophysicalResearch Communications,2010,397:580–585”中公开过,公众可从中国人民解放军军事医学科学院生物工程研究所获得。
下述实施例中所用的Concanavalin A(ConA)是Sigama公司的产品,货号:11028-71-0。
实施例1、抗原及其片段的制备
一、重组质粒的构建
同时构建如下重组质粒:pVAX1-GP、pVAX1-GP△、pVAX1-RBS、pVAX1-RBSP和pVAX1-FU。具体步骤如下:
(一)设计并合成引物
设计用于构建重组质粒pVAX1-GP、pVAX1-GP△、pVAX1-RBS、pVAX1-RBSP和pVAX1-FU的引物,引物序列如表1所示。
表1、用于构建重组质粒的引物序列
GPF | 5’-CCGGAATTCGCCACCATGAAAACTACTTGCTTATTAAT |
GPR | 5’-CCGCTCGAGCTTGTCATCGTCGTCCTTGTAGTCTTAGCCGATGTATTTAGTGAA |
GP△F | 5’-CCGGAATTCGCCACCATGCTGCCAATCCTGGAGATCGC |
GP△R | 5’-CCGCTCGAGTTATTACTTGTCATCGTCGTCCTTGTAGTCATCAGAAGTCCACCACTTGC |
RBSF | 5’-CCGGAATTCGCCACCATGGCTTCTAATATTCAGCCACA |
RBSR | 5’-CCGCTCGAGTTATTACTTGTCATCGTCGTCCTTGTAGTCGTGTCTGTAGCCCTGACC |
RBSP+ | 5’-CCGAGATAATGCAATCAGCACAGCGATAGACAGCAGTAACAGAATGCCGTGTCTGTAGCCCTGACC |
RBSPR | 5’-CCGCTCGAGTTATTACTTGTCATCGTCGTCCTTGTAGTCAGATAATGCAATCAGCACAG |
FUF | 5’-CCGGAATTCGCCACCATGCCAACTACTACTGTTCCAAA |
FUR | 5’-CCGCTCGAGTTATTACTTGTCATCGTCGTCCTTGTAGTCCAGTCTACGCAGACGACA |
(二)GP基因扩增
以pUC57-MGP为模板,以GPF和GPR为引物进行PCR扩增,得到PCR扩增产物1(GP基因)。GP基因的核苷酸序列如序列1所示。
PCR反应体系:pUC57-MGP为模板1μl,2X Q5High-Fidelity DNA Polymerase 25μl,上游引物GPF 2.5μl,下游引物GP R2.5μl,补加灭菌水至50μl。
PCR程序:98℃预变性4分钟;98℃变性10秒,57℃退火30秒,72℃延伸30S,30个循环,最后72℃延伸2min,4℃保温。
(三)GP△、RBS、RBSP、FU基因扩增
1、以pUC57-MGP为模板,以GP△F和GP△R为引物进行PCR扩增,得到PCR扩增产物2(GP△基因)。GP△基因的核苷酸序列如序列1中自5’末端起第55-1944位核苷酸所示。
PCR扩增的退火温度为57℃,72℃延伸10秒。
2、以pUC57-MGP为模板,以RBSF和RBSR为引物进行PCR扩增,得到PCR扩增产物3(RBS基因)。RBS基因如序列1中自5’末端起第73-564位核苷酸所示。
PCR扩增的退火温度为57℃,72℃延伸15秒。
3、以pUC57-MGP为模板,以RBSF和RBSP+为引物进行PCR扩增,得到PCR扩增产物RBSP+,再以PCR扩增产物RBSP+为模板,以RBSF和RBSPR为引物进行PCR扩增,得到PCR扩增产物4(RBSP基因)。RBSP基因的核苷酸序列依次由序列1中自5’末端起第73-564位和第1966-2010位核苷酸分子组成。
PCR扩增的退火温度为60℃,72℃延伸15秒。
4、以pUC57-MGP为模板,以FUF和FUR为引物进行PCR扩增,得到PCR扩增产物5(FU基因)。FU基因如序列1中自5’末端起第1204-1686位核苷酸所示。
PCR扩增的扩增退火温度为58℃,72℃延伸15秒。
(四)用限制性内切酶EcoRI与XhoI分别双酶切PCR扩增产物1、PCR扩增产物2、PCR扩增产物3、PCR扩增产物4和PCR扩增产物5,分别得到GP、GP△、RBS、RBSP和FU基因;用限制性内切酶EcoRI与XhoI双酶切载体pVAX1,得到载体大片段;将GP、GP△、RBS、RBSP和FU基因分别与载体大片段连接,分别得到重组质粒pVAX1-GP,pVAX1-GP△,pVAX1-RBS,pVAX1-RBSP和pVAX1-FU。
1%琼脂糖凝胶分析重组质粒pVAX1-GP△,pVAX1-RBS,pVAX1-RBSP,pVAX1-FU和pVAX1空载体结果如图1所示。并将重组质粒pVAX1-GP,pVAX1-GP△,pVAX1-RBS,pVAX1-RBSP和pVAX1-FU进行测序验证,结果正确。
重组质粒pVAX1-GP表达带有FLAG标签的GP蛋白(氨基酸序列如序列2所示),重组质粒pVAX1-GP△表达带有FLAG标签的GP△蛋白(氨基酸序列依次由序列2第19-648位氨基酸和序列2第682-689位氨基酸组成),重组质粒pVAX1-RBS表达带有FLAG标签的RBS蛋白(氨基酸序列依次由序列2第25-188位氨基酸和序列2第682-689位氨基酸组成),重组质粒pVAX1-RBSP表达带有FLAG标签的RBSP蛋白(氨基酸序列依次由序列2第25-188位氨基酸、序列2第656-670位氨基酸和序列2第682-689位氨基酸组成)和重组质粒pVAX1-FU表达带有FLAG标签的FU蛋白(氨基酸序列依次由序列2第402-562位氨基酸和序列2第682-689位氨基酸组成)。
二、重组蛋白GP的分段表达
(一)重组质粒的构建
1、pET24a(+)-GP1△的构建
以pUC57-MGP为模板,以GP1△F和GP1△R为引物进行PCR扩增,得到GP1△基因(GP1△基因的核苷酸序列为序列1第73-720位所示),用限制性内切酶BamHI与XhoI双酶切GP1△基因,得到GP1△片段;用限制性内切酶BamHI与XhoI双酶切pET24a(+)载体(购自Novagen公司),得到载体大片段;将GP1△基因片段与载体大片段连接,得到重组质粒,将其命名为pET24a(+)-GP1△。
GP1△F:5’-CGCGGATCCATGGCTTCTAATATTCAGCCACAA-3’;
GP1△R:5’-CCGCTCGAGGTGTGCAGTTGGCAGTGGCAG-3’。
2、pET32a(+)-GPM的构建
以pUC57-MGP为模板,以GPMF和GPMR为引物进行PCR扩增,得到GPM基因(GPM基因核苷酸序列为序列1第721-1560位所示),用限制性内切酶BamHI与XhoI双酶切GPM基因,得到GPM基因片段;用限制性内切酶BamHI与XhoI双酶切pET32a(+)载体(购自Novagen公司),得到载体大片段;将GPM基因片段与载体大片段连接,得到重组质粒,将其命名为pET32a(+)-GPM。
GPMF:5’-CGCGGATCCGATGCAACAAAATTAAACACAA-3’;
GPMR:5’-CCGCTCGAGACCAGATACGCAGCTCAGCATCG-3’。
3、pET24a(+)-GP2△的构建
以pUC57-MGP为模板,以GP2△F和GP2△R为引物进行PCR扩增,得到GP2△基因(GP2△基因的核苷酸序列为序列1第1306-1944位所示),用限制性内切酶BamHI与XhoI双酶切GP2△基因,得到GP2△片段;用限制性内切酶BamHI与XhoI双酶切pET24a(+)载体,得到载体大片段;将GP2△基因片段与载体大片段连接,得到重组质粒,将其命名为pET24a(+)-GP2△。
GP2△F:5’-CGCGGATCCAATATCCTGTGGAGAGAGGGC-3’;
GP2△R:5’-CCGCTCGAGATCAGAAGTCCACCACTTGCC-3’。
将重组质粒pET24a(+)-GP1△,pET32a(+)-GPM和pET24a(+)-GP2△进行测序验证,结果正确。重组质粒pET24a(+)-GP1△表达GP1△蛋白,GP1△蛋白的氨基酸序列为序列2第25-240位,重组质粒pET32a(+)-GPM表达GPM蛋白,GPM蛋白的氨基酸序列为序列2第241-520位氨基酸,重组质粒pET24a(+)-GP2△表达GP2△蛋白,GP2△蛋白的氨基酸序列为序列2第436-648位。
(二)重组菌的构建
将重组质粒pET24a(+)-GP1△,pET32a(+)-GPM和pET24a(+)-GP2△分别转化BL21感受态细胞(购自北京天根生化科技有限公司),各取1μl质粒加入相应的感受态细胞中,冰浴30分钟,42℃热激90秒,再冰浴180秒,向感受态中加入900μl无抗性LB培养基,37℃,150rpm复苏45分钟,将复苏pET24a(+)-GP1△、pET24a(+)-GP2△菌液均匀涂布在卡那霉素抗性的LB平板,将复苏pET32a(+)-GPM菌液均匀涂布在氨苄青霉素抗性的LB平板37℃倒置培养16小时,分别得到重组菌。
(三)重组蛋白的诱导表达
挑取重组pET24a(+)-GP1△、pET24a(+)-GP2△菌的单克隆菌落,加入10ml 50μg/ml卡那霉素抗性的LB培养基,37℃,220rpm培养12h;按照体积比1:100接种菌液至1L 50μg/ml卡那霉素抗性的LB培养基,37℃,220rpm培养至OD600=0.5,大约2小时,加入IPTG至终浓度1mM,30℃诱导表达4小时后离心收集GP1△和GP2△重组菌菌体。
挑取重组pET32a(+)-GPM菌的单克隆菌落,加入10ml 100μg/ml氨苄青霉素抗性的LB培养基,37℃,220rpm培养12h;按照体积比1:100接种菌液至1L 100μg/ml氨苄青霉素抗性的LB培养基,37℃,220rpm培养至OD600=0.5,大约2小时,加入IPTG至终浓度1mM,30℃诱导表达4小时后离心收集GPM重组菌菌体。
(四)重组蛋白的纯化
对GP1△、GP2△重组菌菌体和GPM重组菌菌体进行纯化,分别得到纯化后的重组蛋白GP1△,GP2△和GPM。其中,重组蛋白GP1△,GP2△为包涵体形式,GPM为可溶性表达,重组蛋白GP1△和GP2△的具体纯化过程如下:用缓冲液A(50mM Tris-HCl(pH8.0),2mM EDTA,100mM NaCl)漂洗菌体细胞,将漂洗过的菌体细胞悬浮于缓冲液B(50mM Tris-HCl(pH8.0),1mM EDTA,100mM NaCl,1%NP-40)中,冰水中超声破碎(输出比61%,工作5秒,间歇5秒,总时间30分钟)4℃,10000rpm离心30分钟,收集破菌包涵体;将包涵体沉淀用缓冲液Ⅰ(50mMTris-HCl(pH8.0),2mM EDTA,100mM NaCl,0.5%Triton X-100(V/V),4M脲素)、缓冲液Ⅱ(50M Tris-HCl(pH8.0),2mM EDTA,100mM NaCl,3%Triton X-100)、缓冲液Ⅲ(50mM Tris-HCl(pH8.0),2mM EDTA,100mM NaCl,0.5%Triton X-100,2M盐酸胍)分别超声洗涤一次,4℃,10000rpm离心30分钟,收集包涵体沉淀;用含高浓度脲素的缓冲液C(8M脲素,50mM DTT,100mM Tris-HCl(pH8.0),1mM EDTA及脱氧胆酸钠)溶解包涵体。0.45μm滤膜过滤后上Ni亲和柱进行纯化,结合buffer(20mM Na3PO4,500mM NaCl,咪唑30mM,Ph7.4)和洗脱buffer(20mM Na3PO4,500mM NaCl,咪唑500mM,Ph7.4)含8M尿素,将破菌上清、包涵体溶解液上清、目的蛋白洗脱液进行SDS-PAGE电泳,GP1△如图2所示,GP2△如图3所示。
Macrosep超滤离心管(10K)(购自Millipore公司)超滤浓缩目的蛋白洗脱液,分别得到纯化后的大小约为24KD的重组蛋白GP1△和重组蛋白GP2△。
重组蛋白GPM的具体纯化过程如下:结合buffer(20mM Na3PO4,500mM NaCl,咪唑30mM,Ph7.4)重悬GPM重组菌菌体,超声破菌(输出比61%,工作5秒,间歇5秒,总时间30分钟),4℃,10000rpm离心30分钟,收集破菌上清,0.45μm滤膜过滤后上Ni亲和柱进行纯化,收集穿透液、杂蛋白洗脱液和目的蛋白洗脱液。将破菌上清、洗杂峰、洗脱buffer-50洗脱液、洗脱buffer-100洗脱液、洗脱buffer-150洗脱液、洗脱buffer-200洗脱液进行SDS-PAGE电泳,结果如图4所示。
Macrosep超滤离心管(10K)(购自Millipore公司)超滤浓缩目的蛋白洗脱液,得到纯化后的大小约为30kD(因为在pET32a(+)载体中融合了一个肽,导致显示在40KD大小)的重组蛋白GPM。
实施例2、抗原及其片段的免疫活性测定
(一)免疫方法
1、分组
动物选取6周龄Balb/C雌性小鼠,分为生理盐水1组、pVAX1-GP△组、pVAX1-RBS组、pVAX1-RBSP组、pVAX1-FU组、生理盐水2组、BSA蛋白组、GP1△蛋白组、GPM蛋白组、GP2△蛋白组和GP混合蛋白组(GP1△蛋白、GP2△蛋白和GPM蛋白按照质量比1:1:1混合),每组12只小鼠。
2、免疫方式
生理盐水1组、pVAX1-GP△组、pVAX1-RBS组、pVAX1-RBSP组、pVAX1-FU组采取后腿肌肉注射,不加佐剂,2周免疫一次(分别在第0天、第14天和第28天免疫),100μg质粒/只(质粒浓度1mg/ml),生理盐水免疫体积为100μl/只,共3次,免疫前2天注射0.25%盐酸普鲁卡因刺激肌肉。
生理盐水2组、BSA蛋白组、GP1△蛋白组、GPM蛋白组、GP2△蛋白组、GP混合蛋白组采取皮下多点注射,2周免疫一次(分别在第0天、第14天和第28天免疫)。初免35μg蛋白/只(蛋白浓度100μg/ml),生理盐水免疫体积与蛋白组相同,共免疫3次,第二、三次免疫25μg蛋白/只。首次免疫采用弗氏完全佐剂(购自Sigma公司),第二、三次免疫采用弗氏不完全佐剂(购自Sigma公司),均按照质量比1:1混合。
3、采血方式及时间
尾静脉采血,采血时间为免疫前采血1次,每次免疫后第10天采血1次。
(二)血清中抗体滴度测定
1、所采各组小鼠的血液置于室温凝固收缩2小时后,5000rpm离心5分钟,收集上清,即为血清。
2、重组混合蛋白GP(GP1△蛋白、GP2△蛋白和GPM蛋白按照质量比1:1:1混合得到混合蛋白)铺板(Nunc酶标板),2μg/孔,4℃过夜,用PBST(含体积百分含量0.5%吐温-20)溶液洗板4次,每次5分钟;将板用5g/100ml的脱脂牛奶的PBST(含体积百分含量0.5%吐温-20)溶液37℃封闭2小时,PBST(含体积百分含量0.5%吐温-20)溶液洗板4次,每次5分钟;加入步骤1得到的免疫血清,37℃孵育1小时,PBST(含体积百分含量0.5%吐温-20)溶液洗板4次,每次5分钟;加入HRP标记的Goat anti-mouse IgG抗体,37℃孵育1小时,PBST(含体积百分含量0.5%吐温-20)溶液洗板4次,每次5分钟;100μl/孔加入TMB显色液,室温避光反应15分钟,加入2M硫酸终止反应,OD450读数(仪器为Thermo MULTISCAN FC酶标仪)。
生理盐水1组、pVAX1-GP△组、pVAX1-RBS组、pVAX1-RBSP组和pVAX1-FU组血清中抗体滴度结果如图5所示。
生理盐水2组、BSA蛋白组、GP混合蛋白组、GP1△蛋白组、GPM蛋白组和GP2△蛋白组血清中抗体滴度结果如图6所示。
图5和图6表明,无论采取重组质粒免疫还是重组蛋白免疫,GP1△部分抗体滴度很低,GP2△抗体滴度高,与GP混合蛋白组效果类似。
(三)脾淋巴细胞增殖实验
通过分析淋巴细胞增殖水平,可以判断细胞免疫反应水平。
1、于最后一次免疫后,无菌条件下取生理盐水组2、GP混合蛋白组和GP2△蛋白组小鼠的脾脏,按照Mouse 1×Lymphocyte Separation Medium的说明,从达优小鼠淋巴细胞分离液分离小鼠脾淋巴细胞并制备淋巴细胞悬液(重悬于RPMI1640培养基),按照2.5×105Cell/孔铺96孔板,按100μl/孔分别加入重组混合蛋白GP溶液(25μg/ml,溶于RPMI1640培养基)或ConA溶液(10μg/ml,溶于RPMI1640培养基),每组细胞设置4个复孔,将96孔板置于37℃,5%CO2培养箱中培养36小时。
2、按照cell counting kitTetraZ说明,将按10μl/孔加入各细胞孔中,继续培养2小时后,测定OD450,计算刺激指数(SI=刺激孔OD450/对照孔OD450),结果如图7所示。
图7表明,重组混合蛋白GP刺激时,GP2△蛋白组SI低于GP混合蛋白组,表明GP2△蛋白组特异性细胞免疫水平弱于GP混合蛋白组;Con A刺激时,GP2△蛋白组SI高于GP混合蛋白组,表明GP2△蛋白组非特异性细胞免疫状态更为活跃,即抗原GP2△能在一定程度上提高机体的细胞免疫反应水平。
蛋白GP(重组混合蛋白GP)作为特异性刺激源,可刺激淋巴细胞产生特异性增殖反应,抗原GP含较多的T细胞表位,能够诱导更强烈的特异性细胞免疫反应,而抗原GP2△含有的T细胞表位少于GP,诱导的特异性细胞免疫反应弱于抗原GP;ConA是非特异性刺激源,抗原GP2△可能具有某种上调机体淋巴细胞丝裂原受体表达水平的功能,因此在ConA刺激时,抗原GP2△免疫的小鼠脾淋巴细胞增殖反应较抗原GP更为强烈。
(四)假病毒感染中和实验
采用经典的假型病毒的感染中和实验方法(Arnab Basu,et al.JOURNAL OFVIROLOGY,Apr.2011,p.3106–3119;L.Du,et al.Res.Commun.(2010),doi:10.1016/j.bbrc.2010.05.161)验证抗原片段GP2△和FU诱导产生有效中和抗体的能力,为抗原片段GP2△和FU应用于疫苗或中和抗体的开发提供依据。具体步骤如下:
1、假病毒包装:HEK293T细胞以8×105Cell/孔铺6孔板,37℃,5%CO2培养箱培养过夜,至90%融合度。2μg pNL4-3.luc.RE质粒与2μg pVAX1-GP质粒共转染293T细胞,转染试剂lipofectamine3000(购自Invitrogen公司)。转染48小时后,收集培养上清,离心后分装,即为假病毒溶液。
2、假病毒感染中和实验:于实验前一天,按1×104Cell/孔将293T细胞铺于96孔板,待到次日生长至90%融合度。将10μl步骤(二)制备的生理盐水1组、pVAX1-GP△组、pVAX1-FU组、GP2△蛋白组和GP混合蛋白组免疫血清与10μl步骤1制备的假病毒溶液混合于1ml DMEM培养基中,37℃孵育2小时,分别得到混合液。将200μl/孔混合液替换96孔板细胞培养基,每组血清设置4个复孔,将96孔板置于37℃,5%CO2培养箱中培养。24小时后,更换新鲜的含10%FBS的DMEM培养基,继续培养48小时后,按照Britelite plus LuminesenceReporter Gene Assay System说明,使用PerkinElmerEnSpireTM2300MUltiable Reader仪器测定各组发光值,计算假病毒感染抑制率,结果如图8所示。
图8表明,抗原片段GP2△(GP2△蛋白组)、FU(pVAX1-FU组)、GP△(pVAX1-GP△组)与GP(GP混合蛋白组)均能在一定程度上中和HIV-MGP假病毒,其中和能力与盐水组比差异显著,GP2△蛋白组和pVAX1-FU组与GP组中和能力相当。
序列表
<110>中国人民解放军军事医学科学院生物工程研究所
<120>基于马尔堡病毒包膜蛋白的抗原片段、截短体及应用
<160>2
<210>1
<211>2070bp
<212>DNA
<213>人工序列
<220>
<223>
<400>1
atgaaaacta cttgcttatt aatctctctg atcctgattc agggtgtgaa gactctgcca 60
atcctggaga tcgcttctaa tattcagcca caaaacgttg attctgtttg ctctggtaca 120
ctgcagaaga cagaggatgt gcatctgatg ggcttcacac tgtcaggcca gaaggtggct 180
gattctcctc tggaagcttc aaaacgttgg gcattccgtg caggcgttcc ccctaagaac 240
gtggagtaca cagagggtga agaagctaag acatgttata acatttcagt tacagatcca 300
tctggtaagt ctctgctgct ggacccacca acaaatatcc gtgactaccc taaatgtaag 360
actatccatc atatccaggg ccagaaccct catgcacaag gtatcgctct gcacctgtgg 420
ggtgctttct ttttatatga tagaattgct tctacaacta tgtatagagg taaagttttt 480
actgagggta atattgctgc tatgattgtg aataaaacag ttcataaaat gatcttctct 540
cgtcagggtc agggctacag acacatgaat ctgacatcaa caaataaata ctggacatca 600
tctaacggca cacagacaaa tgacactggc tgcttcggca cattacagga atacaattct 660
actaaaaacc agacttgcgc accatctaaa aagccactgc cactgccaac tgcacaccca 720
gaagttaaat taacttcaac ttcaacagat gcaacaaaat taaacacaac tgatccaaat 780
tctgacgacg aagacctgac tacatctggc tctggttctg gtgagcagga gccttacaca 840
acttcagatg cagctactaa gcagggcctg tcatcaacaa tgccacctac tccatcacca 900
cagccttcta ctccacaaca gggtggcaac aacacaaacc actctcaggg cgttgtgaca 960
gaaccaggta aaactaatac aacagctcag ccttctatgc cacctcacaa cactacaaca 1020
atttcaacaa ataacacttc taagcataac ctgtctactc catctgttcc aattcaaaat 1080
gctactaact acaatactca gtctacagca ccagagaacg agcagacatc tgcaccttca 1140
aagacaactt tattacctac tgaaaatcca actacagcta aatctactaa ttcaactaaa 1200
tctccaacta ctactgttcc aaacactact aataagtact caacatcacc atctccaaca 1260
cctaactcta cagctcagca cctggtgtat ttccgtcgta agcgtaatat cctgtggaga 1320
gagggcgata tgttcccttt cctggacggc ctgattaatg cacctattga ttttgatcca 1380
gttccaaaca caaagactat cttcgacgaa tcttcatcat caggcgcttc tgctgaagag 1440
gatcaacatg cttctcctaa tatttcactg acactgtcat attttcctaa agtgaatgag 1500
aacacagctc actctggcga gaacgagaac gattgcgatg ctgagctgcg tatctggtct 1560
gtgcaagaag atgacctggc tgcaggtctg tcttggattc cttttttcgg tcctggcatc 1620
gagggcctgt acacagcagg cttaattaaa aatcagaata atctggtttg tcgtctgcgt 1680
agactggcaa atcagactgc aaaatctctg gaactgctgc tgcgtgttac aactgaggaa 1740
agaacattct ctctgattaa ccgtcatgca attgacttcc tgctggctag atggggcggt 1800
acttgcaaag tgctgggccc agactgctgc atcggcattg aagatctgtc ccgtaacatt 1860
tctgaacaga ttgatcagat taaaaaagat gagcagaaag aaggcactgg ttggggcctg 1920
ggtggcaagt ggtggacttc tgattggggt gtgctgacta acctgggcat tctgttactg 1980
ctgtctatcg ctgtgctgat tgcattatct tgcatttgta gaattttcac taaatacatc 2040
ggcgactaca aggacgacga tgacaagtaa 2070
<210>2
<211>689
<212>PRT
<213>人工序列
<220>
<223>
<400>2
Met Lys Thr Thr Cys Leu Leu Ile Ser Leu Ile Leu Ile Gln Gly Val
1 5 10 15
Lys Thr Leu Pro Ile Leu Glu Ile Ala Ser Asn Ile Gln Pro Gln Asn
20 25 30
Val Asp Ser Val Cys Ser Gly Thr Leu Gln Lys Thr Glu Asp Val His
35 40 45
Leu Met Gly Phe Thr Leu Ser Gly Gln Lys Val Ala Asp Ser Pro Leu
50 55 60
Glu Ala Ser Lys Arg Trp Ala Phe Arg Ala Gly Val Pro Pro Lys Asn
65 70 75 80
Val Glu Tyr Thr Glu Gly Glu Glu Ala Lys Thr Cys Tyr Asn Ile Ser
85 90 95
Val Thr Asp Pro Ser Gly Lys Ser Leu Leu Leu Asp Pro Pro Thr Asn
100 105 110
Ile Arg Asp Tyr Pro Lys Cys Lys Thr Ile His His Ile Gln Gly Gln
115 120 125
Asn Pro His Ala Gln Gly Ile Ala Leu His Leu Trp Gly Ala Phe Phe
130 135 140
Leu Tyr Asp Arg Ile Ala Ser Thr Thr Met Tyr Arg Gly Lys Val Phe
145 150 155 160
Thr Glu Gly Asn Ile Ala Ala Met Ile Val Asn Lys Thr Val His Lys
165 170 175
Met Ile Phe Ser Arg Gln Gly Gln Gly Tyr Arg His Met Asn Leu Thr
180 185 190
Ser Thr Asn Lys Tyr Trp Thr Ser Ser Asn Gly Thr Gln Thr Asn Asp
195 200 205
Thr Gly Cys Phe Gly Thr Leu Gln Glu Tyr Asn Ser Thr Lys Asn Gln
210 215 220
Thr Cys Ala Pro Ser Lys Lys Pro Leu Pro Leu Pro Thr Ala His Pro
225 230 235 240
Glu Val Lys Leu Thr Ser Thr Ser Thr Asp Ala Thr Lys Leu Asn Thr
245 250 255
Thr Asp Pro Asn Ser Asp Asp Glu Asp Leu Thr Thr Ser Gly Ser Gly
260 265 270
Ser Gly Glu Gln Glu Pro Tyr Thr Thr Ser Asp Ala Ala Thr Lys Gln
275 280 285
Gly Leu Ser Ser Thr Met Pro Pro Thr Pro Ser Pro Gln Pro Ser Thr
290 295 300
Pro Gln Gln Gly Gly Asn Asn Thr Asn His Ser Gln Gly Val Val Thr
305 310 315 320
Glu Pro Gly Lys Thr Asn Thr Thr Ala Gln Pro Ser Met Pro Pro His
325 330 335
Asn Thr Thr Thr Ile Ser Thr Asn Asn Thr Ser Lys His Asn Leu Ser
340 345 350
Thr Pro Ser Val Pro Ile Gln Asn Ala Thr Asn Tyr Asn Thr Gln Ser
355 360 365
Thr Ala Pro Glu Asn Glu Gln Thr Ser Ala Pro Ser Lys Thr Thr Leu
370 375 380
Leu Pro Thr Glu Asn Pro Thr Thr Ala Lys Ser Thr Asn Ser Thr Lys
385 390 395 400
Ser Pro Thr Thr Thr Val Pro Asn Thr Thr Asn Lys Tyr Ser Thr Ser
405 410 415
Pro Ser Pro Thr Pro Asn Ser Thr Ala Gln His Leu Val Tyr Phe Arg
420 425 430
Arg Lys Arg Asn Ile Leu Trp Arg Glu Gly Asp Met Phe Pro Phe Leu
435 440 445
Asp Gly Leu Ile Asn Ala Pro Ile Asp Phe Asp Pro Val Pro Asn Thr
450 455 460
Lys Thr Ile Phe Asp Glu Ser Ser Ser Ser Gly Ala Ser Ala Glu Glu
465 470 475 480
Asp Gln His Ala Ser Pro Asn Ile Ser Leu Thr Leu Ser Tyr Phe Pro
485 490 495
Lys Val Asn Glu Asn Thr Ala His Ser Gly Glu Asn Glu Asn Asp Cys
500 505 510
Asp Ala Glu Leu Arg Ile Trp Ser Val Gln Glu Asp Asp Leu Ala Ala
515 520 525
Gly Leu Ser Trp Ile Pro Phe Phe Gly Pro Gly Ile Glu Gly Leu Tyr
530 535 540
Thr Ala Gly Leu Ile Lys Asn Gln Asn Asn Leu Val Cys Arg Leu Arg
545 550 555 560
Arg Leu Ala Asn Gln Thr Ala Lys Ser Leu Glu Leu Leu Leu Arg Val
565 570 575
Thr Thr Glu Glu Arg Thr Phe Ser Leu Ile Asn Arg His Ala Ile Asp
580 585 590
Phe Leu Leu Ala Arg Trp Gly Gly Thr Cys Lys Val Leu Gly Pro Asp
595 600 605
Cys Cys Ile Gly Ile Glu Asp Leu Ser Arg Asn Ile Ser Glu Gln Ile
610 615 620
Asp Gln Ile Lys Lys Asp Glu Gln Lys Glu Gly Thr Gly Trp Gly Leu
625 630 635 640
Gly Gly Lys Trp Trp Thr Ser Asp Trp Gly Val Leu Thr Asn Leu Gly
645 650 655
Ile Leu Leu Leu Leu Ser Ile Ala Val Leu Ile Ala Leu Ser Cys Ile
660 665 670
Cys Arg Ile Phe Thr Lys Tyr Ile Gly Asp Tyr Lys Asp Asp Asp Asp
675 680 685
Lys
Claims (10)
1.如下M1)或M2)或M3)所示的蛋白质:
M1)马尔堡病毒的GP蛋白自N端起第436-648位氨基酸;
M2)马尔堡病毒的GP蛋白自N端起第402-562位氨基酸;
M3)马尔堡病毒的GP蛋白自N端起第19-648位氨基酸。
2.一种蛋白质或其截短体,所述截短体为如下(1)-(5)中任一所示:
(1)氨基酸序列是序列2第436-648位所示的蛋白质;
(2)氨基酸序列是序列2第402-562位所示的蛋白质;
(3)在序列2第436-648位或序列2第402-562位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
(4)将序列2第436-648位或序列2第402-562位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质;
(5)与序列2第436-648位或序列2第402-562位所示的氨基酸序列具有90%以上的同一性且具有相同功能的蛋白质;
所述蛋白质为如下(6)-(9)中任一所示:
(6)氨基酸序列是序列2第19-648位所示的蛋白质;
(7)在序列2第19-648位所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
(8)将序列2第19-648位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质;
(9)与序列2第436-648位或序列2第402-562位所示的氨基酸序列具有90%以上的同一性且具有相同功能的蛋白质。
3.权利要求1所述蛋白质或权利要求2所述蛋白质或其截短体的编码基因。
4.根据权利要求3所述的编码基因,其特征在于:
权利要求1中M1)或M2)所述蛋白质或权利要求2中所述截短体的编码基因为如下1)-4)中任一所示:
1)序列1中自5’末端起第1306位至第1944位核苷酸所示的DNA分子;
2)序列1中自5’末端起第1204位至第1686位核苷酸所示的DNA分子;
3)在严格条件下与1)或2)限定的DNA分子杂交且编码权利要求1中M1)或M2)所述蛋白质或权利要求2中所述截短体的DNA分子;
4)与1)或2)或3)限定的DNA分子具有90%以上的同一性且编码权利要求1中M1)或M2)所述蛋白质或权利要求2中所述截短体的DNA分子;
权利要求1中M3)所述蛋白质或权利要求2中所述蛋白质的编码基因为如下5)-7)任一所示:
5)序列1中自5’末端起第55-1944位核苷酸所示的DNA分子;
6)在严格条件下与5)限定的DNA分子杂交且编码权利要求1中M3)所述蛋白质或权利要求2中所述蛋白质的DNA分子;
7)与5)或6)限定的DNA分子具有90%以上的同一性且编码权利要求1中M3)所述蛋白质或权利要求2中所述蛋白质的DNA分子。
5.下述(a1)-(a4)中的任一种生物材料:
(a1)含有权利要求3或4所述编码基因的表达盒;
(a2)含有权利要求3或4所述编码基因的重组载体;
(a3)含有权利要求3或4所述编码基因的重组菌;
(a4)含有权利要求3或4所述编码基因的转基因细胞系。
6.权利要求1所述蛋白质和/或权利要求2所述蛋白质或其截短体和/或权利要求3或4所述编码基因和/或权利要求5所述的生物材料在制备免疫原和/或抗原中的应用;
或,所述免疫原或抗原是针对马尔堡病毒或马尔堡病毒包膜蛋白GP的。
7.权利要求1所述蛋白质和/或权利要求2所述蛋白质或其截短体和/或权利要求3或4所述编码基因和/或权利要求5所述的生物材料作为抗原在制备针对马尔堡病毒的抗体中的应用;
或,权利要求1所述蛋白质和/或权利要求2所述蛋白质或其截短体和/或权利要求3或4所述编码基因和/或权利要求5所述的生物材料在制备预防和/或治疗马尔堡病毒引起的疾病的产品中的应用;
或,权利要求1所述蛋白质和/或权利要求2所述蛋白质或其截短体和/或权利要求3或4所述编码基因和/或权利要求5所述的生物材料在预防和/或治疗马尔堡病毒引起的疾病中的应用。
8.权利要求1所述蛋白质和/或权利要求2所述蛋白质或其截短体和/或权利要求3或4所述编码基因和/或权利要求5所述的生物材料在如下a1)-a6)中任一种中的应用:
a1)抑制病毒感染;
a2)制备抑制病毒感染的产品;
a3)抑制病毒与宿主细胞结合;
a4)制备抑制病毒与宿主细胞结合的产品;
a5)抑制病毒入侵;
a6)制备抑制病毒入侵的产品;
或,所述病毒为马尔堡病毒。
9.一种产品,其活性成分为权利要求1所述蛋白质或权利要求2所述蛋白质或其截短体;
所述产品的功能为如下b1)-b3)中任一种:
b1)抑制病毒感染;
b2)抑制病毒与宿主细胞结合;
b3)抑制病毒入侵;
或,所述产品为药物或疫苗;
或,所述病毒为马尔堡病毒。
10.一种多抗,是以权利要求1所述蛋白质或权利要求2所述蛋白质或其截短体为免疫原制备得到的。
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CN108715856A (zh) * | 2018-05-07 | 2018-10-30 | 中国人民解放军军事科学院军事医学研究院 | 一种以人复制缺陷腺病毒为载体的马尔堡病毒病疫苗 |
CN108715856B (zh) * | 2018-05-07 | 2019-08-20 | 中国人民解放军军事科学院军事医学研究院 | 一种以人复制缺陷腺病毒为载体的马尔堡病毒病疫苗 |
WO2019214110A1 (zh) * | 2018-05-07 | 2019-11-14 | 中国人民解放军军事科学院军事医学研究院 | 一种以人复制缺陷腺病毒为载体的马尔堡病毒病疫苗 |
US11453704B2 (en) | 2018-05-07 | 2022-09-27 | Academy Of Military Medical Science, Pla | Recombinant human replication-deficient adenovirus comprising a modified nucleic acid encoding the Marburg virus envelope glycoprotein |
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