CN115043947A - 一种克里米亚-刚果出血热病毒Zera-Gn蛋白纳米颗粒、制备方法及其用途 - Google Patents
一种克里米亚-刚果出血热病毒Zera-Gn蛋白纳米颗粒、制备方法及其用途 Download PDFInfo
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Abstract
本发明涉及疫苗技术领域,涉及一种克里米亚‑刚果出血热病毒的Gn蛋白,添加Zera序列并使用杆状病毒表达载体表达制备得到蛋白纳米颗粒,所述Zera‑Gn蛋白纳米颗粒制备方法简单,免疫小鼠后能够产生高效的免疫保护作用,对于防控克里米亚‑刚果出血热具有良好的应用前景。
Description
技术领域
本申请涉及疫苗技术领域,特别是涉及一种Zera序列与克里米亚-刚果出血热病毒Gn蛋白融合而成的纳米颗粒疫苗的研制方法及其应用。
背景技术
克里米亚-刚果出血热(Crimean-Congo hemorrhagic fever,CCHF)是由克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus,CCHFV)引起的一种蜱传人畜共患病,主要通过蜱虫、草食家畜和宠物等途径传播给人类,目前没有针对CCHFV的有效疫苗,该病致死率约为20%-30%,临床症状与流感症状相似,包括发热、腹泻、疲倦以及嗜睡等特征,在严重病例中,患者会出现肾脏病变,肝功能衰竭和肺损伤甚至死亡。近年来,该病有进一步扩散的趋势,其潜在的流行性、高死亡率、院内感染以及治疗和预防的困难,CCHF将对我们公共卫生安全造成安全威胁。
CCHFV属于布尼亚病毒科内罗病毒属,与布尼亚病毒科的其他成员一样,由三段单负链RNA片段组成,分别为L(12kb)、M(6.8kb)和S(3kb),L片段编码RNA依赖性RNA聚合酶、M片段编码结构糖蛋白(Gn和Gc)和S片段编码核衣壳蛋白。其中M片段具有遗传多样性,但在许多菌株中其抗原表位是保守的,近年来,M片段编码的Gn和Gc抗原中和表位已被确定,Gn和Gc均为I型整合跨膜蛋白,含有很多糖基化修饰位点以及78-80个半胱氨酸残基,形成大量二硫键和复杂的二级结构,现已表明,CCHFV Gn结构糖蛋白可引发机体强烈的病毒中和反应,对克里米亚-刚果出血热诊断和疫苗研究具有重要意义,也是研制亚单位疫苗的理想靶抗原。
杆状病毒表达***是目前真核蛋白表达应用最广泛的表达***之一,具有磷酸化、糖基化、乙酰化和形成二硫键等翻译后修饰机制,杆状病毒制备过程操作简单,易于扩大培养,高产率等优点,并且杆状病毒本身具有很强的免疫佐剂活性。
Zera序列来源于γ-玉米醇溶蛋白(protein body,PB)的N端,富含脯氨酸结构域,可在多种不同的宿主中表达,包括杆状病毒表达***。当与目标蛋白融合表达时,Zera序列可提高目标蛋白的表达量并将目标蛋白包裹形成致密的球形蛋白体,定位于内质网中,避免被细胞质中的蛋白酶水解,易于纯化,并且Zera蛋白具有很强的佐剂活性,能诱导机体产生强烈的免疫反应。
发明内容
为了解决上述问题,本申请一方面提供一种Zera-Gn蛋白纳米颗粒,包括克里米亚-刚果出血热病毒(CCHFV)Gn序列和Zera标签。
在一些实施方式中,所述Zera-Gn蛋白纳米颗粒C端添加His6标签。
在一些实施方式中,所述Zera-Gn蛋白纳米颗粒氨基酸序列如SEQ ID NO:2所示。
另一方面,本申请提供一种核苷酸序列,其编码所述Zera-Gn蛋白纳米颗粒。
在一些实施方式中,编码所述Zera-Gn蛋白纳米颗粒的核苷酸序列如SEQ ID NO:1所示。
另一方面,本申请提供一种重组表达载体。
在一些实施方式中,所述重组表达载体携带有SEQ ID NO:1所示的核酸序列,能够表达Zera-Gn蛋白纳米颗粒。
在一些实施方式中,所述重组表达载体类型为pFastBac-Dual载体。
另一方面,本申请提供一种Zera-Gn蛋白纳米颗粒的制备方法,包括以下步骤:
(1)构建表达Zera-Gn蛋白纳米颗粒的重组质粒;
(2)转化重组质粒转至大肠杆菌感受态细胞中筛选;
(3)提取阳性转化子中的重组质粒,转染真核细胞;
(4)培养细胞后,收获细胞,裂解得到Zera-Gn蛋白纳米颗粒。
在一些实施方式中,所述步骤(2)转化方式为热激转化、电转化、脂质体转化等方式中的一种。
在一些实施方式中,所述步骤(2)筛选为抗性筛选、蓝白斑筛选以及底物筛选等方式中的一种。
在一些实施方式中,所述步骤(4)中,分离获得Zera-Gn蛋白纳米颗粒的方法为密度梯度离心,例如蔗糖密度梯度离心。
另一方面,本申请提供一种克里米亚-刚果出血热(CCHF)疫苗,其含有所述Zera-Gn蛋白纳米颗粒。
在一些实施方式中,所述CCHF疫苗包括佐剂。
另一方面,本申请提供一种工程化细胞,其表达所述Zera-Gn蛋白纳米颗粒,或含有所述编码所述Zera-Gn蛋白纳米颗粒的核苷酸序列,或含有所述表达Zera-Gn蛋白纳米颗粒的重组表达载体。
另一方面,本申请提供所述Zera-Gn蛋白纳米颗粒,或编码所述Zera-Gn蛋白纳米颗粒的核苷酸序列,或所述表达Zera-Gn蛋白纳米颗粒的重组表达载体在制备预防和/或治疗CCHF的药物中的应用。
发明的效果
本申请所提供的候选疫苗具有以下优点:
1、所述Zera-Gn蛋白纳米颗粒制备方法简单,安全性高,可通过蔗糖密度梯度离心获得目的蛋白纳米颗粒。
2、所述Zera-Gn蛋白纳米颗粒免疫原性高,在免疫小鼠后,能产生强烈的体液和/或细胞免疫反应,表明此蛋白纳米颗粒疫苗免疫后能引发小鼠体内产生特异性的免疫效应。
附图说明
图1:pFastBac-Dual-Zera-Gn重组载体构建示意图。
图2:重组Zera-Gn蛋白纳米颗粒的Western Blot鉴定。
图3:重组Zera-Gn蛋白纳米颗粒的电镜照片图。
图4:重组Zera-Gn蛋白纳米颗粒免疫小鼠后血清ELISA检测结果。
图5:重组Zera-Gn蛋白纳米颗粒免疫小鼠后脾淋巴细胞指数检测结果。
图6:重组Zera-Gn蛋白纳米颗粒免疫小鼠后血清中细胞因子ELISA检测结果。
具体实施方式
为使本发明的技术方案和有益效果能够更加明显易懂,下面通过列举具体实施例的方式进行详细说明。其中,附图不一定是按比例绘制的,局部特征可以被放大或缩小,以更加清楚的显示局部特征的细节;除非另有定义,本文所使用的技术和科学术语与本申请所属的技术领域中的技术和科学术语的含义相同。
本发明所述技方案提及的一些试剂配方:
PBS:8gNaCl,0.2g KCl,1.44g Na2HPO4,0.24g KH2PO4溶于ddH2O定容至1L;
抗原包被缓冲液:0.85M Na2CO3/NaHCO3缓冲液;
ELISA洗涤液:0.05%Tween-20的PBS溶液;
ELISA封闭液:5%BSA的PBS溶液;
ELISA终止液:2M H2SO4。
实施例1:目的基因的获取及表达载体的构建
选择克里米亚-刚果出血热中国新疆株HANM-18(GenBank:MN832722.1)Gn序列,融合Zera序列(GenBank:KU593570.1),并根据昆虫杆状病毒***对Zera-Gn基因序列进行密码子优化,得到如SEQ ID NO:1所示的目的基因。
用Sph I和Xho I酶切位点将目的基因构建至pFastBac-Dual载体(Invirogen,美国)p10端。得到pFastBac-Dual-Zera-Gn重组载体,载体图谱如图1所示。
实施例2:Zera-Gn蛋白纳米颗粒表达重组杆状病毒的包装、表达检测与种毒的制备
将上述获得的重组质粒pFastBac-Dual-Zera-Gn和pFastBac-Dual空载质粒分别转化入DH10Bac感受态细胞中,经过蓝白斑筛选出阳性克隆。提取阳性杆状病毒质粒,通过TransIT-LT1(Mirus公司)试剂分别转染入密度约为1×106的Sf9细胞中,96h后吹打细胞,离心收集细胞上清,分别命名为Zera-Gn(氨基酸序列如SEQ ID NO:2所示)和rvAc-dual。以Anti-Gn和Anti-6×His为一抗,通过Western Blot方法检测重组蛋白的表达情况,结果如图2所示。
实施例3:重组杆状病毒的扩增、Zera-Gn蛋白纳米颗粒的纯化与检测
按照MOI=0.1将P1代重组杆状病毒接种Sf9细胞中进行病毒扩增,连续扩增两代后得到P3代重组病毒。将P3代病毒以MOI=0.5剂量接种摇瓶培养的悬浮H5细胞,接种后96h通过低速离心收集细胞。裂解细胞,通过密度梯度离心分离Zera-Gn蛋白纳米颗粒和其他杂质,收集40%-60%蔗糖之间的浑浊带,进一步超速离心去除蔗糖,最后用少量PBS溶解。将蛋白纳米颗粒负载到200目铜网上,磷钨酸染色后在电镜下观察形态,如图3所示。
实施例4:小鼠免疫实验
将24只6-8周龄的雌性BALB/c小鼠随机分成以下3组:①PBS组;②rvAc-dual组;③Zera-Gn纳米疫苗组,每组8只。采用皮下多点免疫途径,分别在0、14和28d进行免疫。其中PBS组每只小鼠注射PBS 200μL;病毒组每只小鼠免疫1×107PFUrvAc-dual;实验组每只小鼠免疫10μg Zera-Gn蛋白纳米颗粒。分别在第0d、14d和28d进行眼球取血,在第35d、42d分别对每组4只小鼠进行眼球取血与脾淋巴细胞增殖实验。
实施例5:抗体水平检测
为了评估小鼠对于各种抗原刺激所产生的体液免疫效应,采用间接ELISA法,分别用经缓冲液稀释后浓度为1μg/mL的Gn蛋白包被,ELISA封闭液封闭,分别孵育0d、14d、28d、35d和42d五个时间段采集的血清(抗体稀释液1:100稀释),再孵育二抗(Proteintech,SA00001-2),加显色液显色,其中每两步间均用ELISA洗涤液洗板3次,每次2min。最后加入ELISA终止液终止显色反应,检测450nm波长下的OD值。结果如图4所示,其中***表示P<0.001。重组病毒免疫组的小鼠血清针对Gn抗原均能表现出显著的特异性IgG反应著,表明Zera-Gn蛋白纳米颗粒免疫小鼠后在其体内具有良好的免疫性能。
实施例6:脾淋巴细胞增殖实验
为了评估小鼠对于抗原刺激所产生的细胞免疫效应,使用小鼠淋巴细胞分离液(北京达科为生物技术有限公司)分离35d和42d的小鼠脾淋巴细胞,具体操作流程见产品说明书。计数分离后的各组脾淋巴细胞,加100μL于96孔板,终浓度稀释至2×105cell/mL。待细胞贴壁后,每孔分别加入100μL稀释后的抗原(2μg)、1640培养基和阳性对照(刀豆蛋白,终浓度10μg/mL)。CO2培养箱恒温培养42h后每孔加入20μL MTT(5mg/mL),继续培养4h,弃尽细胞培养上清液,每孔加入100μL DMSO溶液,振荡器上震荡1min,检测490nm波长下的OD值,并计算刺激指数(SI=刺激孔OD值/未刺激孔OD)。结果如图5所示,其中NS表示无显著性差异,***表示P<0.001。Zera-Gn蛋白纳米颗粒组的小鼠相比较于其他对照组,对于相应抗原刺激能表现出较高的刺激指数,表明Zera-Gn蛋白纳米颗粒免疫后能引发小鼠体内产生特异性的细胞免疫效应。
实例7:血清细胞因子水平分析
用商品化ELISA试剂盒对0d和42d的免疫小鼠血清进行检测,通过分析IL-4和TNF-α水平的变化来反映免疫小鼠的细胞免疫应答情况,结果如图6所示。Zera-Gn蛋白纳米颗粒第42d血清中的IL-4和TNF-α水平均显著高于0d的所有血清抗体水平,以及42d的PBS、rvAc-dual组血清抗体水平(P<0.001)。Zera-Gn蛋白纳米颗粒42d的免疫小鼠血清中IL-4与TNF-α的含量分别为49.697±2.732pg/mL,21.974±1.159pg/mL,结果表明Zera-Gn蛋白纳米颗粒可以作为免疫原诱导机体产生显著的细胞免疫反应。
本申请序列信息如下:
>SEQ ID NO:1
atgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccacctccacgcatacaagcggcggctgcggctgccagccaccgccgccggttcatctaccgccgccggtgcatctgccacctccggttcacctgccacctccggtgcatctcccaccgccggtccacctgccgccgccggtccacctgccaccgccggtccatgtgccgccgccggttcatctgccgccgccaccatgccactaccctactcaaccgccccggcctcagcctcatccccagccacacccatgcccgtgccaacagccgcatccaagcccgtgccagaccccatggtcagaggaatctggaggtgactgtatctcacgtacccaactattaagaacggaaatagccgaaatacactcagacaactacggtggaccgggtgataaaatcacaatttgcaacggttccacgattgtagatcagcgcttggggtcagagttggggtgctataccatcaatagggtgaagtcctttaaactatgtgagaactccgcaataggcaagagttgcgagatagactcgacgcccgtcaaatgccgccagggcttttgtctgaagataacgcaggaaggccggggtcacgtaaagttaagccgggggtcggaaattgttttggacgcctgcgattcgagctgtgagattatgatcccacgaggcacaggagacatacttgttgattgcagtggaggccagcaacacttcttaaaggataatttgatcgatctgggatgtcctaatatccccttactcggtaagatggcgatctacatttgcaggatgtcaaaccatcccaaaaccacgatggcctttttgttctggttttcgttcgggtacgttattacctgtatactttgtaaagtgattttttacctgctaattgtagctgggaccgtcgggaaaaaattcaaacaatatagggagttaaaaccgcagacgtgtacaatatgcgaaaccacgccggtcaatgctattgacgcggaaatgcatgatcttaattgcagttacaatatttgcccatactgcgcatctcgcttgacatcgtacgatctggcacgacatgtcatgcagtgtcccaaacgcaaggagaaaatcgaggagaccgaactatatctgaatctcgaacgcatcccttgggtcgttcatcatcaccaccatcac
>SEQ ID NO:2
MRVLLVALALLALAASATSTHTSGGCGCQPPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHVPPPVHLPPPPCHYPTQPPRPQPHPQPHPCPCQQPHPSPCQTPWSEESGGDCISRTQLLRTEIAEIHSDNYGGPGDKITICNGSTIVDQRLGSELGCYTINRVKSFKLCENSAIGKSCEIDSTPVKCRQGFCLKITQEGRGHVKLSRGSEIVLDACDSSCEIMIPRGTGDILVDCSGGQQHFLKDNLIDLGCPNIPLLGKMAIYICRMSNHPKTTMAFLFWFSFGYVITCILCKVIFYLLIVAGTVGKKFKQYRELKPQTCTICETTPVNAIDAEMHDLNCSYNICPYCASRLTSYDLARHVMQCPKRKEKIEETELYLNLERIPWVVHHHHHH
应当理解,以上实施例均为示例性的,不用于包含权利要求所包含的所有可能的实施方式。在不脱离本公开范围的情况下,还可以在以上实施例的基础上做出各种变形和改变。同样的,也可以对以上实施例的各个技术特征进行任意组合,以形成可能没有被明确描述的本发明的另外的实施例。因此,上述实施例仅表达了本发明的几种实施方式,不对本发明专利的保护范围进行限制。
序列表
<110> 宁夏大学
<120> 一种克里米亚-刚果出血热病毒Zera-Gn蛋白纳米颗粒、制备方法及其用途
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1215
<212> DNA
<213> Artificial Sequence
<400> 1
atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg 60
catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120
catctgccac ctccggttca cctgccacct ccggtgcatc tcccaccgcc ggtccacctg 180
ccgccgccgg tccacctgcc accgccggtc catgtgccgc cgccggttca tctgccgccg 240
ccaccatgcc actaccctac tcaaccgccc cggcctcagc ctcatcccca gccacaccca 300
tgcccgtgcc aacagccgca tccaagcccg tgccagaccc catggtcaga ggaatctgga 360
ggtgactgta tctcacgtac ccaactatta agaacggaaa tagccgaaat acactcagac 420
aactacggtg gaccgggtga taaaatcaca atttgcaacg gttccacgat tgtagatcag 480
cgcttggggt cagagttggg gtgctatacc atcaataggg tgaagtcctt taaactatgt 540
gagaactccg caataggcaa gagttgcgag atagactcga cgcccgtcaa atgccgccag 600
ggcttttgtc tgaagataac gcaggaaggc cggggtcacg taaagttaag ccgggggtcg 660
gaaattgttt tggacgcctg cgattcgagc tgtgagatta tgatcccacg aggcacagga 720
gacatacttg ttgattgcag tggaggccag caacacttct taaaggataa tttgatcgat 780
ctgggatgtc ctaatatccc cttactcggt aagatggcga tctacatttg caggatgtca 840
aaccatccca aaaccacgat ggcctttttg ttctggtttt cgttcgggta cgttattacc 900
tgtatacttt gtaaagtgat tttttacctg ctaattgtag ctgggaccgt cgggaaaaaa 960
ttcaaacaat atagggagtt aaaaccgcag acgtgtacaa tatgcgaaac cacgccggtc 1020
aatgctattg acgcggaaat gcatgatctt aattgcagtt acaatatttg cccatactgc 1080
gcatctcgct tgacatcgta cgatctggca cgacatgtca tgcagtgtcc caaacgcaag 1140
gagaaaatcg aggagaccga actatatctg aatctcgaac gcatcccttg ggtcgttcat 1200
catcaccacc atcac 1215
<210> 2
<211> 405
<212> PRT
<213> Artificial Sequence
<400> 2
Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser
1 5 10 15
Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro
20 25 30
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu
35 40 45
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val
50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro
65 70 75 80
Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro His Pro
85 90 95
Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro Ser Pro Cys Gln
100 105 110
Thr Pro Trp Ser Glu Glu Ser Gly Gly Asp Cys Ile Ser Arg Thr Gln
115 120 125
Leu Leu Arg Thr Glu Ile Ala Glu Ile His Ser Asp Asn Tyr Gly Gly
130 135 140
Pro Gly Asp Lys Ile Thr Ile Cys Asn Gly Ser Thr Ile Val Asp Gln
145 150 155 160
Arg Leu Gly Ser Glu Leu Gly Cys Tyr Thr Ile Asn Arg Val Lys Ser
165 170 175
Phe Lys Leu Cys Glu Asn Ser Ala Ile Gly Lys Ser Cys Glu Ile Asp
180 185 190
Ser Thr Pro Val Lys Cys Arg Gln Gly Phe Cys Leu Lys Ile Thr Gln
195 200 205
Glu Gly Arg Gly His Val Lys Leu Ser Arg Gly Ser Glu Ile Val Leu
210 215 220
Asp Ala Cys Asp Ser Ser Cys Glu Ile Met Ile Pro Arg Gly Thr Gly
225 230 235 240
Asp Ile Leu Val Asp Cys Ser Gly Gly Gln Gln His Phe Leu Lys Asp
245 250 255
Asn Leu Ile Asp Leu Gly Cys Pro Asn Ile Pro Leu Leu Gly Lys Met
260 265 270
Ala Ile Tyr Ile Cys Arg Met Ser Asn His Pro Lys Thr Thr Met Ala
275 280 285
Phe Leu Phe Trp Phe Ser Phe Gly Tyr Val Ile Thr Cys Ile Leu Cys
290 295 300
Lys Val Ile Phe Tyr Leu Leu Ile Val Ala Gly Thr Val Gly Lys Lys
305 310 315 320
Phe Lys Gln Tyr Arg Glu Leu Lys Pro Gln Thr Cys Thr Ile Cys Glu
325 330 335
Thr Thr Pro Val Asn Ala Ile Asp Ala Glu Met His Asp Leu Asn Cys
340 345 350
Ser Tyr Asn Ile Cys Pro Tyr Cys Ala Ser Arg Leu Thr Ser Tyr Asp
355 360 365
Leu Ala Arg His Val Met Gln Cys Pro Lys Arg Lys Glu Lys Ile Glu
370 375 380
Glu Thr Glu Leu Tyr Leu Asn Leu Glu Arg Ile Pro Trp Val Val His
385 390 395 400
His His His His His
405
Claims (9)
1.一种Zera-Gn蛋白纳米颗粒,其特征在于,所述Zera-Gn蛋白纳米颗粒氨基酸序列如SEQ ID NO:2所示。
2.编码权利要求1所述Zera-Gn蛋白纳米颗粒的核苷酸片段。
3.如权利要求2所述的核苷酸片段,其特征在于,具有如SEQ ID NO:1所示的序列。
4.一种重组表达载体,其特征在于,含有权利要求2-3任一项所述的核苷酸片段。
5.如权利要求4所述的重组表达载体,其特征在于,其载体类型为pFastBac-Dual载体。
6.一种工程化细胞,其具备以下特征中的一项或多项:
(1)表达权利要求1所述Zera-Gn蛋白纳米颗粒;
(2)含有权利要求2-3任一项所述核苷酸片段;
(3)含有权利要求4-5任一项所述重组表达载体。
7.制备权利要求1所述的Zera-Gn蛋白纳米颗粒的方法,包括以下步骤:
(1)构建表达Zera-Gn蛋白纳米颗粒的重组质粒;
(2)转化重组质粒转至大肠杆菌感受态细胞中筛选;
(3)提取阳性转化子中的重组质粒,转染真核细胞;
(4)培养细胞后,收获细胞,裂解得到Zera-Gn蛋白纳米颗粒。
8.权利要求1所述Zera-Gn蛋白纳米颗粒,或权利要求2-3任一项所述核苷酸片段,或权利要求4-5所述重组表达载体,或权利要求6所述工程化细胞在制备预防和/或治疗克里米亚-刚果出血热药物或疫苗中的应用。
9.一种疫苗,其含有权利要求1所述Zera-Gn蛋白纳米颗粒,或权利要求2-3任一项所述核苷酸片段的表达产物,或权利要求4-5任一项所述重组表达载体的表达产物,或权利要求6所述工程化细胞表达产物。
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