CN114163503B - 两种表达水痘-带状疱疹病毒的gE蛋白的重组腺病毒及应用 - Google Patents
两种表达水痘-带状疱疹病毒的gE蛋白的重组腺病毒及应用 Download PDFInfo
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Abstract
本发明提供了两种表达水痘‑带状疱疹病毒的gE蛋白的重组腺病毒及应用,涉及生物医药技术领域。本发明提供了两种基于水痘‑带状疱疹病毒gE蛋白的核苷酸序列制备的重组腺病毒,以可表达水痘‑带状疱疹病毒gE蛋白的重组腺病毒同源或异源组合作为带状疱疹病毒疫苗,其在小鼠上能在短时间内诱导机体产生强烈的细胞及体液免疫反应。
Description
技术领域
本发明属于生物医药技术领域,具体涉及两种表达水痘-带状疱疹病毒的gE蛋白的重组腺病毒及应用。
背景技术
人是水痘-带状疱疹病毒(人疱疹病毒3型)(Varicella-Zoster Virus,VZV)的唯一自然宿主,其感染对象主要是儿童,几乎所有儿童在其成长过程中均感染过VZV,儿童感染后的临床表现为水痘,痊愈后少量病毒会长期潜伏在脊髓后根神经节或颅神经的感觉神经节内,当机体免疫力降低、免疫功能受损或年龄相关的免疫衰减时,残留在神经节内的病毒会大量复制并沿感觉神经传播至其所支配区域的皮肤组织复制,引发神经损伤和带状疱疹(Herpes zoster)。带状疱疹主要见于中老年人群,由于随着年龄增长,宿主体内针对VZV的细胞免疫相应降低,导致带状疱疹发病率随着年龄的增长而增加,60岁人群发病率为6~8/1000人/年,80岁人群发病率升至12/1000人/年。
目前,用于带状疱疹治疗的药物主要有口服药阿昔洛韦和泛昔洛韦,以及局部外用药喷昔洛韦,但这些药物并不能清除病毒,只能暂时抑制病毒复制,当宿主机体免疫力降低时,病毒会再次复制。因此,接种疫苗成为预防带状疱疹最有效的手段。迄今为止,美国食品药品监督管理局(FDA)先后批准了默沙东公司研发的减毒活疫苗-Zostavax和葛兰素史克公司(GSK)研制的亚单位疫苗-Shingrix用于预防50岁及以上人群带状疱疹。研究显示,这两种疫苗均能诱导机体产生细胞免疫和体液免疫,显着降低老年人带状疱疹和带状疱疹后神经痛的发生率。接种减毒活疫苗-Zostavax后,带状疱疹发生率可降低51.3%,带状疱疹后神经痛的发生率可降低66.5%,但其对带状疱疹的预防效果会随着年龄的增长而下降。由于减毒活疫苗存在病毒潜伏及诱发带状疱疹等风险,美国已于2020年7月1日停止销售减毒活疫苗-Zostavax。接种亚单位疫苗-Shingrix后,50岁以上人群总体保护效果高达97.2%,且不随年龄的增长而降低。2020年6月,该疫苗(欣安立适)在中国上市,尽管其在国内市场供不应求,但其高昂的费用提醒我们亟需开发拥有自主知识产权的带状疱疹疫苗。
发明内容
有鉴于此,本发明的目的在于提供两种表达水痘-带状疱疹病毒的gE蛋白的重组腺病毒及应用,以可表达带状疱疹病毒gE蛋白的重组腺病毒同源或异源组合作为状疱疹病毒疫苗,能在短时间内诱导机体产生强烈的细胞及体液免疫反应。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种水痘-带状疱疹病毒gE蛋白,所述gE蛋白的核苷酸序列如SEQID NO.1所示。
本发明还提供了两种包含上述gE蛋白的核苷酸序列的重组腺病毒,所述两种重组腺病毒分别以人26型复制缺陷型重组腺病毒pAd26和猩猩63型复制缺陷型重组腺病毒pChAd63为骨架载体。
本发明还提供了上述重组腺病毒的构建方法,包括以下步骤:构建含有上述gE蛋白的核苷酸序列的穿梭质粒载体pShuttle26/gE和pShuttle63/gE;
将所述穿梭质粒载体pShuttle26/gE与pAd26进行DNA组装,得人26型复制缺陷型重组腺病毒质粒;
将所述穿梭质粒载体pShuttle63/gE与pChAd63进行DNA组装,得猩猩63型复制缺陷型重组腺病毒质粒;
分别将所述人26型复制缺陷型重组腺病毒质粒和猩猩63型复制缺陷型重组腺病毒质粒酶切后转染宿主细胞,拯救后分别得所述人26型重组腺病毒和猩猩63型重组腺病毒。
优选的,所述穿梭质粒载体pShuttle26/gE和pShuttle63/gE的构建方法,包括:分别将上述gE蛋白的核苷酸序列插入pSH和pShuttle63的SalI和KpnI酶切位点处,得pShuttle26/gE和pShuttle63/gE。
本发明还提供了上述重组腺病毒制备带状疱疹病毒疫苗中的应用。
优选的,所述带状疱疹病毒疫苗的剂型包括注射剂、滴鼻剂或喷雾剂。
优选的,所述带状疱疹病毒疫苗中,所述重组腺病毒以同源或异源的方式进行组合应用。
本发明还提供了一种带状疱疹病毒疫苗,所述疫苗以上述重组腺病毒为有效成分。
优选的,以人26型重组腺病毒为初免组合物和加强组合物;
或以猩猩63型重组腺病毒为初免组合物和加强组合物。
优选的,以人26型重组腺病毒为初免组合物,以猩猩63型重组腺病毒为加强组合物;
或以猩猩63型重组腺病毒为初免组合物,以人26型重组腺病毒为加强组合物pShuttle26/gE。
有益效果:本发明提供了一种密码子优化的水痘-带状疱疹病毒gE蛋白核苷酸序列,并基于所述gE蛋白的核苷酸序列制备了重组腺病毒,以可表达状疱疹病毒gE蛋白的重组腺病毒同源或异源组合作为带状疱疹病毒疫苗,其在小鼠上能在短时间内诱导机体产生强烈的细胞及体液免疫反应。
附图说明
图1为本发明实施例所述的重组腺病毒质粒酶切鉴定;左侧图为pAd26/gE的质粒酶切鉴定,其中M:DL 15000Marker;1:BstZ17I单酶切;2:SbfI单酶切;3:PacI和SpeI双酶切;右侧图为pChAd63/gE质粒酶切鉴定,其中M:DL 15000Marker;1:NheI单酶切;2:NdeI单酶切;3:BstZ17I单酶切;
图2为本发明实施例所述的gE蛋白表达鉴定;
图3为本发明实施例所述的血清IgG1和血清IgG2a;
图4为本发明实施例所述的细胞免疫。
具体实施方式
本发明提供了一种密码子优化的水痘-带状疱疹病毒gE蛋白核苷酸序列,所述gE蛋白的核苷酸序列如SEQ ID NO.1所示。
本发明SEQ ID NO.1所示的核苷酸序列为对gE(NC_001348.1)进行密码子优化后得到的序列,经所述密码子优化后显著提高gE蛋白表达水平。
本发明还提供了两种包含上述gE蛋白的核苷酸序列的重组腺病毒,所述两种重组腺病毒分别以人26型复制缺陷型重组腺病毒pAd26和猩猩63型复制缺陷型重组腺病毒pChAd63为骨架载体。
本发明所述pAd26和pChAd63已在中国专利CN202010863762.X进行过公开,在此不再赘述。
本发明还提供了上述重组腺病毒的构建方法,包括以下步骤:构建含有上述gE蛋白的核苷酸序列的穿梭质粒载体pShuttle26/gE和pShuttle63/gE;
将所述穿梭质粒载体pShuttle26/gE与pAd26进行DNA组装,得人26型复制缺陷型重组腺病毒质粒;
将所述穿梭质粒载体pShuttle63/gE与pChAd63进行DNA组装,得猩猩63型复制缺陷型重组腺病毒质粒;
分别将所述人26型复制缺陷型重组腺病毒质粒和猩猩63型复制缺陷型重组腺病毒质粒酶切后转染宿主细胞,拯救后分别得所述人26型重组腺病毒和猩猩63型重组腺病毒。
本发明所述穿梭质粒载体的构建方法,优选包括:分别将上述gE蛋白的核苷酸序列插入pShuttle26和pShuttle63的SalI和KpnI酶切位点处,得穿梭质粒pShuttle26/gE和pShuttle63/gE。本发明对所述插入的方式并没有特殊限定,优选利用双酶切法,具体的用限制性核酸内切酶SalI和KpnI分别双酶切pShuttle26和pShuttle63,同时由于SEQ IDNO.1所示的核苷酸序列两端分别包括SalI和KpnI的粘性末端,将回收的载体片段和目的片段连接后转化DH10B感受态细菌,提质粒后得穿梭质粒载体pShuttle26/gE和pShuttle63/gE。本发明所述pShuttle26和pShuttle63已在中国专利CN202010863762.X进行过公开,在此不再赘述。
本发明将所述pShuttle26/gE与人26型腺病毒骨架载体(pAd26)进行DNAAssembly(DNA组装),得人26型重组腺病毒质粒(pAd26/gE);将所述穿梭质粒载体pShuttle63/gE与猩猩63型腺病毒骨架载体(pChAd63)进行DNA组装,得猩猩63型重组腺病毒质粒(pChAd63/gE)。本发明对所述DNAAssembly的方法并没有特殊限定,优选采用试剂盒法。
得重组腺病毒质粒pAd26/gE和pChAd63/gE后,本发明将所述重组腺病毒质粒酶切后转染宿主细胞,拯救后得所述重组腺病毒。本发明优选利用相同的酶对所述重组腺病毒质粒进行酶切,所述酶切所用的酶优选包括PacI和SpeI,而后利用乙醇沉淀回收线性化重组腺病毒DNA基因组。本发明所述转染优选包括利用Lipofectamine 2000将所得线性化重组腺病毒DNA基因组分别转染到293细胞,拯救获得表达带状疱疹gE蛋白的重组腺病毒rAd26/gE和rChAd63/gE。
本发明在得到的所述重组腺病毒后,优选还包括进行扩大培养、纯化和表达验证,更优选的包括取纯化后重组腺病毒病毒rAd26/gE和rChAd63/gE各2μl,分别接种于293细胞,80%细胞出现病变后收集细胞,利用gE蛋白单抗进行Dotblot和Western blot检测重组腺病毒蛋白表达情况,结果显示rAd26/gE和rChAd63/gE均表达gE蛋白。
本发明还提供了上述重组腺病毒的同源或异源组合在制备带状疱疹病毒疫苗中的应用。
本发明实施例中,所述疫苗以人26型复制缺陷腺病毒和猩猩63型复制缺陷型腺病毒为载体,携带经密码子优化的gE蛋白,所述疫苗在小鼠体内具有良好的免疫原性,能在短时间内诱导机体产生强烈的细胞及体液免疫反应,因此可用于制备疫苗。
本发明对所述带状疱疹病毒疫苗的剂型并没有特殊限定,优选包括注射剂、滴鼻剂或喷雾剂,实施例中以注射剂为例进行说明,但是不能仅将其认定为本发明的全部保护范围。
本发明还提供了一种带状疱疹病毒疫苗,所述疫苗以上述重组腺病毒为有效成分。
本发明所述疫苗优选包括初免组合物和加强组合物,所述初免组合物和加强组合物可相同或不同,如以人26型重组腺病毒为初免组合物和加强组合物;或以猩猩63型重组腺病毒为初免组合物和加强组合物;或以人26型重组腺病毒为初免组合物,以猩猩63型重组腺病毒为加强组合物;或以猩猩63型重组腺病毒为初免组合物,以人26型重组腺病毒为加强组合物。
本发明所述疫苗优选为注射剂,将腺病毒溶解在10mM Tris-HCl中,终浓度为109PFU/mL。
下面结合实施例对本发明提供的一种水痘-带状疱疹病毒的gE蛋白、重组腺病毒及应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
重组腺病毒质粒的构建和鉴定
一、穿梭质粒的构建
用限制性核酸内切酶SalI和KpnI分别双酶切pShuttle26和人工合成的gE核苷酸序列,凝胶电泳回收载体片段和目的片段,连接、转化感受态细菌DH10B,获得pShuttle26/gE。
用限制性核酸内切酶SalI和KpnI分别双酶切pShuttle63和人工合成的gE核苷酸序列,凝胶电泳回收载体片段和目的片段,连接、转化感受态细菌DH10B,获得pShuttle63/gE。
二、重组腺病毒质粒的构建
用限制性核酸内切酶BamHI和MluI双酶切pShuttle26/gE,凝胶电泳回收目的片段,用限制性核酸内切酶PacI单酶切pAd26,虾碱性磷酸酶(rSAP)去磷酸化后无水乙醇沉淀回收载体片段。运用HiFi DNA Assembly试剂盒的方法,组装载体片段和目的片段,转化感受态细菌DH10B,获得重组腺病毒质粒pAd26/gE。
用限制性核酸内切酶ScaI和AseI双酶切pShuttle63/gE,凝胶电泳回收目的片段,用限制性核酸内切酶HpaI单酶切pChAd63,用虾碱性磷酸酶(rSAP)去磷酸化,再用无水乙醇沉淀回收载体片段。运用HiFi DNA Assembly试剂盒的方法,组装载体片段和目的片段,转化感受态细菌DH10B,获得重组腺病毒质粒pChAd63/gE。
实施例2
重组腺病毒的拯救及表达鉴定
用限制性核酸内切酶PacI和SpeI酶切重组腺病毒质粒pAd26/gE,乙醇沉淀回收。用Lipofectamine 2000将所得线性化重组腺病毒DNA基因组分别转染到293细胞,拯救获得表达带状疱疹gE蛋白的重组腺病毒rAd26/gE;用限制性核酸内切酶PacI酶切重组腺病毒质粒pChAd63/gE,乙醇沉淀回收。用Lipofectamine 2000将所得线性化重组腺病毒DNA基因组分别转染到293细胞,拯救获得表达带状疱疹gE蛋白的重组腺病毒rChAd63/gE。
取纯化后重组腺病毒病毒rAd26/gE和rChAd63/gE各2μl,分别接种于293细胞,80%细胞出现病变后收集细胞,利用gE蛋白单抗进行Dot blot和Western blot检测重组腺病毒蛋白表达情况,结果如图2所示,rAd26/gE和rChAd63/gE均表达gE蛋白。
实施例3
带状疱疹疫苗的初免-加强免疫
一、动物免疫
6-8周龄的雌性C57BL/6小鼠,分成5组,第0d,采集基础血清,第1d,第一次肌肉注射免疫;在免疫后第21d肌肉注射第二次免疫,两次肌肉注射剂量相同(Shingrix免疫剂量为5μg/50μl/鼠,rAd26/gE和rChAd63/gE免疫剂量为108PFU/100μl/鼠)。第28d,处死采集脾淋巴细胞和免疫后血清。
分组及处理如下:
第一组(G1组):阴性对照(PBS初免-PBS加强组);
第二组(G2组):阳性对照(Shingrix初免-Shingrix加强组);
第三组(G3组):rAd26/gE初免-rAd26/gE加强组;
第四组(G4组):rChAd63/gE初免-rChAd63/gE加强组;
第五组(G4组):rAd26/gE初免-rChAd63/gE加强组。
二、免疫后小鼠血清IgG1和IgG2a检测
纯化gE蛋白按40ng/孔包被酶标板、ELISA方法检测小鼠血清抗体IgG1和IgG2a,结果如图3所示,重组腺病毒免疫小鼠后,诱导产生Th1/Th2平衡的血清抗体。
三、免疫后小鼠细胞免疫效果分析
利用ELISPOT的方法分析细胞免疫效果,计数每3×105个脾细胞中经gE蛋白和gE多肽分别刺激后分泌IFN-γ和IL-2的脾淋巴细胞数量,结果如图4所示,重组腺病毒免疫小鼠后,诱导产生较高的细胞免疫。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京交通大学
<120> 两种表达水痘-带状疱疹病毒的gE蛋白的重组腺病毒及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1872
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggcaccg tgaacaagcc tgtggttgga gtgctcatgg gctttggcat catcacaggc 60
accctgagga tcaccaatcc tgtgcgggct tctgtgctga gatacgacga ctttcacatc 120
gacgaggaca aactggacac caacagcgtg tacgagcctt actaccacag cgaccacgcc 180
gaatcgtcct gggtgaacag aggcgagtct agcagaaagg cctatgatca caactctcct 240
tacatctggc ctagaaacga ctacgacggc ttcctggaaa acgcccacga acaccacgga 300
gtatacaacc agggcagagg catcgatagc ggagaacggc tgatgcagcc cacccagatg 360
agcgcccagg aggatctggg cgacgatacc ggcattcacg tgatccccac cctgaatggc 420
gatgacagac acaagatcgt gaatgtggac caaagacagt acggcgacgt gttcaagggc 480
gacctgaacc ctaagcccca aggccagcgc ctcatcgagg tgtcagtgga agagaaccat 540
cccttcaccc tgagagcccc aatccagcgg atctacggcg tgcgatacac agagacatgg 600
tccttcctgc ccagccttac atgtacagga gatgccgccc cagccatcca acacatctgc 660
ctgaagcaca caacctgctt ccaagacgtc gtcgtggacg tggactgcgc tgagaatacc 720
aaggaagatc agctggccga gatcagctac agattccagg gcaagaaaga agccgatcag 780
ccttggatcg tggtgaacac ctctaccctg ttcgatgagc tggagctgga ccctccggaa 840
atcgagcctg gcgtgctgaa agtgttgcgg accgaaaaac agtatctggg cgtgtacatc 900
tggaacatgc ggggctctga tggcacctcc acatacgcta cattcctggt cacctggaag 960
ggcgacgaga agactcggaa ccccacccca gccgtgaccc cccagcccag aggcgccgag 1020
ttccacatgt ggaattacca tagccacgtg ttttctgtgg gcgacacctt tagcctggcc 1080
atgcacctgc agtataagat ccacgaggcc cctttcgacc tgctgctgga atggctgtac 1140
gtccctatcg atcctacctg ccagcccatg cggctgtatt ctacatgcct gtaccaccct 1200
aacgcccctc agtgcctgtc tcacatgaac agcggctgca ccttcacaag ccctcacctg 1260
gctcagagag tggccagcac agtgtaccag aactgtgaac acgccgacaa ctacaccgcc 1320
tactgcctgg gaatcagcca tatggaacct agcttcggcc tgatcctgca cgacggtgga 1380
accacactga agttcgtgga tacacccgag agcctgagcg gcctgtacgt gttcgtggtg 1440
tacttcaacg ggcacgtcga ggccgtggcc tacaccgtgg tgtccaccgt tgatcacttc 1500
gtgaacgcta tcgaggaaag aggatttcct ccaacagctg gccagcctcc agcaacaacc 1560
aagcccaagg aaattacccc tgttaacccc ggcaccagcc ccctgctgag atacgctgcc 1620
tggaccggcg gcctggccgc cgtggtgctg ttatgtctgg tcatcttcct gatctgtacc 1680
gccaagcgga tgagagtgaa ggcctaccgg gtggacaaaa gcccttataa ccagagcatg 1740
tactacgccg gcctgcctgt ggacgatttc gaggacagcg agagcaccga caccgaggaa 1800
gagttcggca atgccatcgg aggcagccac ggcggaagct cctacacagt gtacatcgac 1860
aagacaagat ga 1872
Claims (3)
1.两种包含水痘-带状疱疹病毒gE蛋白的核苷酸序列的重组腺病毒在制备带状疱疹病毒疫苗中的应用,其特征在于,所述gE蛋白的核苷酸序列如SEQ ID NO.1所示;所述两种重组腺病毒分别以人26型复制缺陷型重组腺病毒pAd26和猩猩63型复制缺陷型重组腺病毒pChAd63为骨架载体;
所述带状疱疹病毒疫苗中,所述重组腺病毒以同源或异源的方式进行组合应用。
2.根据权利要求1所述应用,其特征在于,所述带状疱疹病毒疫苗的剂型包括注射剂、滴鼻剂或喷雾剂。
3.一种带状疱疹病毒疫苗,其特征在于,所述疫苗以两种包含水痘-带状疱疹病毒gE蛋白的核苷酸序列的重组腺病毒为有效成分;
所述gE蛋白的核苷酸序列如SEQ ID NO.1所示;所述两种重组腺病毒分别以人26型复制缺陷型重组腺病毒pAd26和猩猩63型复制缺陷型重组腺病毒pChAd63为骨架载体;
以人26型重组腺病毒为初免组合物和加强组合物;或以猩猩63型重组腺病毒为初免组合物和加强组合物;
或以人26型重组腺病毒为初免组合物,以猩猩63型重组腺病毒为加强组合物;或以猩猩63型重组腺病毒为初免组合物,以人26型重组腺病毒为加强组合物。
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