CN111701016A - 逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用 - Google Patents
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Abstract
本发明公开一种逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用,针对特定个体或特定人群感染的原病毒序列,设计亚单位疫苗、灭活疫苗、减活疫苗、重组疫苗和核酸疫苗等。能够直接激活人体免疫系统产生特异性抗患者所携带逆转录病毒的抗体,既回避了流行中逆转录病毒病毒株耐药的痼疾,又特异性靶向患者所携带逆转录病毒毒株。由于逆转录病毒感染者所携带的原病毒序列是整合入人体基因组的DNA序列,它相较于其他感染形式中的RNA序列具有高度保守性,故据此应用设计开发的疫苗具有更强的特异性,是克服目前逆转录病毒疫苗研发瓶颈的一个候选者,具有更好的应用前景。
Description
技术领域
本发明属于医药技术领域,具体涉及逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用。
背景技术
逆转录病毒(Retrovirus)又称反转录病毒,为单链正义RNA包膜病毒。逆转录病毒科主要包括RNA肿瘤病毒亚科、慢病毒亚科和泡沫病毒亚科三类病毒。RNA肿瘤病毒亚科中常见的病毒种类有白血病病毒、肉瘤病毒、乳腺瘤病毒、人类噬T淋巴细胞病毒等。慢病毒亚科中常见的病毒种类有人免疫缺陷病毒、绵羊脱髓鞘性脑白质炎病毒、马传染性贫血病毒等。泡沫病毒亚科中常见的病毒种类是猫、牛、人泡沫病毒。
逆转录病毒通常为球形病毒,其衣壳为20面体的立体对称结构,病毒外覆盖包膜。逆转录病毒基因组中含有逆转录酶基因和整合酶基因。其生活周期中重要的特征为逆转录和基因组整合。具体为该病毒感染宿主细胞后,利用自身编码的逆转录酶使病毒基因组RNA转变为双链DNA,然后病毒的整合酶将病毒DNA连接到宿主细胞基因组中,此时逆转录病毒DNA又称为原病毒(provirus)。逆转录病毒基因组整合在宿主染色体上的位点是随机的,而这个过程是逆转录病毒复制病毒遗传物质的必经阶段。宿主细胞会将病毒DNA作为自身基因组的一部分,翻译和转录病毒基因,产生组装病毒所需的蛋白质,从而包装出新的病毒,成熟的病毒会以芽生(budding)的方式释放。逆转录病毒通常会在宿主细胞中长期持续性感染。
逆转录病毒家族中,对人类公共卫生造成严重影响的以人免疫缺陷病毒、人类噬T淋巴细胞病毒、内源性逆转录病毒为最。
人免疫缺陷病毒(Human immunodeficiency virus,HIV)属于逆转录病毒科慢病毒亚科,是导致获得性免疫缺陷综合征(艾滋病,Acquired Immune Deficiency Syndrome,AIDS)的病毒。HIV通过破坏人体的T淋巴细胞阻断人体细胞免疫和体液免疫过程,导致免疫系统瘫痪,致使人体出现各类微生物感染或肿瘤等并发症,引发艾滋病。由于HIV具有变异极其迅速等特点,至今仍无有效的疫苗,并且虽然治疗药物种类颇多,但仍无法实现治愈,HIV感染对人类健康构成极大威胁。
HIV颗粒的直径约为100nm,具有包膜,核衣壳为二十面体对称结构,内含有单股正链RNA基因组的两个副本,为二倍体。病毒最外层蛋白为包膜表面糖蛋白gp120和跨膜糖蛋白gp41。再往内层则是基质蛋白,定位于病毒包膜与病毒衣壳蛋白中间。在病毒衣壳蛋白内包含复制过程必不可少的病毒蛋白:逆转录酶,蛋白酶和整合酶。病毒表面糖蛋白gp120具有五个可变区和几个恒定区。病毒受体CD4结合结构域位于恒定区,而共受体(也称为辅助受体或辅受体)的结合域在可变区3(V3环),这个区也是病毒变异极为频繁的区域。gp41镶嵌在包膜中,并介导感染过程中病毒包膜与细胞质膜之间的融合,相比gp120变异较少。根据对共受体的偏好性,HIV分为T淋巴细胞嗜性(X4,偏好CXCR4),巨噬细胞嗜性(R5,偏好CCR5)和双嗜性(X4/R5)。
HIV的表面糖蛋白gp120与跨膜糖蛋白gp41在病毒感染细胞时起到重要作用,它们以非共价方式连接,在病毒颗粒表面以多聚体(常为三聚体)的形式存在。细胞受体为CD4分子,共受体为趋化因子受体CXCR4或CCR5。受体和共受体主要位于CD4+T淋巴细胞、单核细胞/巨噬细胞谱系的细胞,以及其他一些靶细胞,如朗格汉斯细胞、树突细胞和某些脑细胞的质膜上。在病毒感染靶细胞过程中,首先是gp120与CD4分子结合,形成gp120-CD4复合体,在此复合体中gp120构象发生变化从而与共受体CCR5或CXCR4结合,该结合使得gp120与gp41分离,进一步诱导跨膜糖蛋白gp41的构象发生改变,从而使gp41上的融合多肽暴露。随后,融合多肽插入到靶细胞膜中,使病毒包膜与细胞膜拉近直至最后融为一体。该膜融合过程导致病毒核衣壳进入细胞质中。
HIV的核衣壳进入细胞后,将会脱壳释放出其基因组RNA,从而进入复制阶段。该RNA被逆转录酶反转录成互补DNA,并进一步转化成双链DNA,整个过程被称为逆转录。这也是逆转录病毒的重要特征之一。HIV逆转录酶在复制时出错率很高,导致每逆转录一次病毒RNA,则有3-4个新的突变被引入转录后的DNA产物中,突变体的基因组在感染过程中快速地积累,最终在同一感染宿主中形成一系列具有许多核苷酸差异的病毒准种。这些突变的病毒有些具有了逃脱体内免疫系统控制的能力,有些产生了对现有HIV药物的抗性,对HIV疫苗和药物研发的造成了巨大的阻碍。
在病毒因子和宿主细胞因子的帮助下,逆转录过程形成的病毒基因组双链DNA被包装成为整合前复合体,该复合体进入细胞核并利用整合酶整合到宿主细胞染色体中,整合位点的选择是随机的,但优先选择转录活跃的基因。整合状态的病毒基因组称为原病毒,在宿主细胞核内呈潜伏状态,随细胞分裂进入子代细胞。只要感染细胞不断分裂,病毒基因组就不断被复制。在一定的条件下,潜伏的原病毒被激活,开始进行转录。除了调控和辅助蛋白之外,所有的逆转录病毒的蛋白均是以多蛋白前体形式进行初始翻译,随后被蛋白酶水解为单个蛋白。病毒蛋白被修饰加工后,与病毒基因组RNA组装,然后通过含有病毒包膜蛋白gp120/gp41复合物的细胞膜区域出芽,从而形成完整的新HIV病毒颗粒,进而感染其他细胞。
人类噬T淋巴细胞病毒(Human T-cell lymphotropic virus,HTLV)属于逆转录病毒科慢病毒亚科,是致瘤性RNA病毒。这种病毒主要分为4种亚型,其中1型(HTLV-1)和2型(HTLV-2)为主要类型,3型(HTLV-3)和4型病毒(HTLV-4)于2005年在西非喀麦隆被发现。这四种亚型具有60-70%的序列同源性。
HTLV颗粒呈球形,直径约为100nm。外面有包膜糖蛋白gp46,膜上嵌有跨膜蛋白gp21。病毒颗粒内含有正二十面体结构的核衣壳,由衣壳蛋白p24组成,内含有核壳蛋白p15以及两条单链RNA基因组。基因组全长约为8507个碱基对。HTLV的生活周期同许多逆转录病毒相似。均经过以下阶段:吸附、膜融合、进入、脱壳、逆转录、病毒整合到宿主基因组中、适当条件下转录翻译、合成蛋白质、组装和出芽释放病毒。它还有一个重要特征是能编码亮氨酸拉链蛋白因子,该因子由一条互补的负链RNA所编码,行使负调控病毒复制和增强病毒持续感染的功能。HTLV-1可引起成人T细胞白血病(adult T-cell leukemia,ATL)、HTLV-1相关性脊髓病/热带痉挛性截瘫(HAM/TSP)、葡萄膜炎、HTLV-1相关的感染性皮炎、淋巴腺炎等。HTLV-2的致病性较弱,可引起T-多毛细胞/巨粒细胞白血病。HTLV-3和HTLV-4的致病性还未明确。
内源性逆转录病毒(Endogenous retrovirus,ERV)是整合在基因组中的逆转录病毒基因序列,通常被认为是很久之前由外源性逆转录病毒对宿主细胞的感染和整合而形成的。这种病毒整合基因序列通过孟德尔遗传方式被遗传下来,持续存在于宿主后代基因组中。ERV是逆转座子的一种,可以通过转座作用增加其在基因组中的拷贝。在人和小鼠基因组中,ERV可以占到8%和10%。
逆转录病毒的复制周期需要将病毒的遗传物质整合到宿主细胞的核基因组中。绝大多数的逆转录病毒都只感染体细胞,极偶然的情况下它们会感染生殖细胞(或者产生卵子和精子的细胞)。更少的情况下,这种整合了逆转录病毒的生殖细胞会发育成为可以繁殖的个体,因此一般认为该逆转录病毒仅整合到了生物体的种系细胞中。这种生物体就将长久携带插入的逆转录病毒基因,并将其作为自身基因组的一个部分,这就成为了内源性逆转录病毒。ERV可以像等位基因一样向后代遗传。许多ERV已经在宿主的基因组中延续了数百万年。
ERV本身作为一种重复序列在宿主基因组中大量存在,使得宿主细胞逐渐进化出一种机制来抑制ERV的表达,尤其是在胚胎发育的早期阶段。如果这种调控抑制机制失衡,ERV异常高表达就会引发肿瘤的发生。在人类多种肿瘤组织中都发现了HERV的高表达,而电离辐射、紫外线和DNA损伤化合物都可能诱导ERV的表达。HERV-K家族成员可以编码两个癌蛋白Rec和Np9,这两种蛋白可以和抑癌基因PLZF的蛋白产物相互作用,促进细胞转化成瘤。而且,ERV可以作为顺式元件调控插入位点附近的宿主基因的正常表达,可以激活癌基因、抑制抑癌基因的表达,从而诱发肿瘤。基于这些特性,一些HERV可以被用作肿瘤诊断或预后的生物标志物。比如HERV-K家族成员表达的Gag蛋白,其抗体水平就与前列腺患者分级和生存率显著相关。同时,靶向HERV的肿瘤免疫疗法也在被积极研究中。
综上所述,逆转录病毒家族许多成员均可导致广泛而且严重的公共卫生问题,其疫苗研发具有巨大市场需求。
由于逆转录病毒家族有整合进入宿主基因组的特性,而整合进入宿主基因组的原病毒序列为双链DNA,具有稳定性,因此,基于原病毒序列设计针对逆转录病毒的疫苗,相对于基于地区流行株设计的逆转录病毒疫苗,有相对优越性;另外,多重耐药株(multidrugresistant(MDR)strain)以及多重耐药株的二重感染(superinfections)和体内重组,给传统鸡尾酒疗法(“高效抗逆转录病毒治疗”,Highly Active Antiroviral Therapy(HAART))带来巨大挑战,对HIV传播的防控和艾滋病患者的生命健康带来巨大威胁。因此,HIV相关疫苗的开发,尤其针对保守原病毒序列的疫苗开发,成为未来逆转录病毒防治的重点研发方向。
发明内容
本发明目的在于提供一种逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用,将逆转录病毒原病毒序列应用于制备逆转录病毒疫苗中,以弥补逆转录病毒耐药株不断出现给现有抗病毒疗法的挑战,以及毒株差异给传统疫苗研发带来的困扰,在逆转录病毒疫苗方面有重要应用前景。
为实现上述目的,本发明采用以下技术方案:
一种逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用,针对特定个体或特定人群感染的原病毒序列,设计制备亚单位疫苗、灭活疫苗、减活疫苗、重组疫苗和核酸疫苗等。
进一步地,所述原病毒序列包括逆转录病毒全病毒序列,或病毒某一个或某几个蛋白序列,或相应蛋白的结构域。
进一步地,所述核酸疫苗包括DNA疫苗和RNA疫苗。
进一步地,所述逆转录病毒包括RNA肿瘤病毒亚科、慢病毒亚科和泡沫病毒亚科等家族中的所有成员。
进一步地,所述逆转录病毒疫苗的接种途径包括皮内注射、皮下注射、肌内注射、口服等。
更进一步地,所述逆转录病毒疫苗应用于人、猫、牛等物种逆转录病毒感染的非治疗目的应用和预防中。
与现有技术相比,本发明具有以下有益效果:
本发明提供一种逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用,所述逆转录病毒疫苗设计的核心在于针对特定个体或特定人群感染的原病毒序列,而非常见流行株序列,设计亚单位疫苗、灭活疫苗、减活疫苗、重组疫苗和核酸疫苗等。能够直接激活人体免疫系统产生特异性抗患者所携带逆转录病毒的抗体,既回避了流行中逆转录病毒病毒株耐药的痼疾,又特异性靶向患者所携带逆转录病毒毒株。逆转录病毒疫苗既可以基于逆转录病毒感染者全病毒序列或病毒某一个或某几个蛋白序列或蛋白结构域的个体化定制,也可以是针对特定人群(某逆转录病毒)中某强致病病毒株(isolate)中某一个或某几个蛋白或是蛋白结构域的广谱设计。由于逆转录病毒感染者所携带的原病毒序列是整合入人体基因组的DNA序列,它相较于其他感染形式中的RNA序列具有高度保守性,故据此设计开发的疫苗具有更强的特异性,是克服目前逆转录病毒疫苗研发瓶颈的一个候选者,具有更好的应用前景。
附图说明
图1为本发明逆转录病毒疫苗设计概括图;
图2为本发明实施例中HIV原病毒检测引物及探针设计图;
图3为本发明实施例中HIV原病毒疫苗设计图(ALVAC-HIVpro)。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。
以下实施例以HIV病毒为例,但本发明提供的逆转录病毒原病毒序列在制备逆转录病毒疫苗中的应用适用于全部逆转录病毒,而不仅限于在HIV疫苗研发中的应用;实施例中所涉及试剂及磁珠仅为相应等同物(equivalents)的一个例证,本发明意在涵盖所有等同物。
实施例
1、获取病人特异性完整可感染性原病毒(intact proviral DNA)序列。
1.1、CD4阳性(CD4+)T淋巴细胞的获取。外周血单核细胞(peripheral bloodmononuclear cells,PBMCs),依照厂家说明书,采用Ficoll-Paque PLUS(GE HealthcareLife Sciences)试剂盒经密度梯度离心后获得。之后将得到的外周血单核细胞采用EasySep Human CD4+T-Cell Enrichment Kit(StemCell Technologies)试剂盒经免疫磁珠筛选后可得到总CD4阳性T淋巴细胞。
1.2、由于部分HIV原病毒可潜伏于静息态CD69阴性、CD25阴性、HLA-DR阴性(CD4+,CD69–,CD25–and HLA-DR–)的三阴性CD4阳性T淋巴细胞(以后以“静息态三阴性CD4阳性T淋巴细胞”指代)。为了获取该部分细胞中HIV原病毒的序列信息,这些静息态三阴性CD4阳性T淋巴细胞可采用磁珠分选法分离,即分别采用Miltenyi Biotec.公司的CD25-Biotin、CD69MicroBead Kit II、Anti–HLA-DR MicroBeads磁珠经三次亲和后得到。以上获得的静息态三阴性CD4阳性T淋巴细胞经流式细胞术确认阳性率达到95%以上为合格。
1.3、获取病人特异性完整可感染性原病毒序列(intact proviral DNA assay,IPDA)。首先,取大约500万CD4阳性T淋巴细胞,采用天根血液基因组抽提试剂盒提取全基因组DNA(抽提过程中注意小心操作以避免基因组过度碎片化),并采用分光光度计(NanoDrop2000,Thermo)测定其DNA浓度。之后,采用巢式PCR鉴定所整合HIV基因组的完整性及分型(引物见附录):其中使用巢式PCR的外侧引物(outer primers)可以基本确定HIV原病毒基因组的完整性,而巢式PCR的内侧引物(inner primers,扩增条件A、B)可以用于确定HIV原病毒基因组的类型和突变情况。以上PCR扩增条件均针对HIV HXB2株优化。其中,巢式PCR扩增条件A主要针对HIV原病毒基因组两端的包装信号(the packaging signal,Ψ),因为如果HIV原病毒基因组发生缺失突变,大概率会出现在这里。巢式PCR的内侧引物(innerprimers)左侧反应扩增的Ψ区域主要覆盖HXB2参考基因组的692–797位核苷酸。如果所检测的HIV原病毒基因组是完整的,那么5′6-FAM-标记的水解探针会被活化并产生蓝色荧光信号。PCR扩增条件B主要针对HIV原病毒基因组的逆转录蛋白响应元件(Rev-responseelement,RRE),位于HXB2参考基因组的7736–7851位核苷酸。该反应含有两种探针,一种为5′端特异性针对野生型HIV原病毒基因组中RRE序列的VIC标记探针;另一种为针对经APOBEC-3G诱导高度变异后的HIV原病毒基因组相应序列的未标记序列,在这里,根据PCR反应能否产生荧光信号即可判断该HIV原病毒基因组是否为含有野生型RRE的HIV原病毒。如果巢式PCR反应同时检测到了FAM荧光信号和VIC荧光信号,说明该HIV原病毒基因组可视为完整野生型病毒;如果巢式PCR反应只检测到了FAM荧光信号,说明该HIV原病毒基因组在RRE序列存在突变,归类于3′缺陷病毒;如果巢式PCR反应只检测到了VIC荧光信号,说明该HIV原病毒基因组在Ψ序列存在突变,归类于5′缺陷病毒;如果巢式PCR反应未检测到任何荧光信号,则说明该反应液所含全基因组DNA未整合HIV原病毒或属于极少数(大约3.8%)在RRE序列和Ψ序列同时存在突变的HIV原病毒。
HIV原病毒检测引物及探针序列(基于HIV-1HXB2株参考序列设计)如下表1所示,HIV-1HXB2株参考序列如SEQ ID NO:10所示。
表1
1.4、HIV原病毒基因组的相对定量。由于病人体内可能整合了不同的HIV原病毒,而且每种HIV原病毒的整合数量、其疫苗效价可能不一致,这可能需要根据不同HIV原病毒及其相对整合数量调整疫苗中不同疫苗成分的含量,因此可能涉及对病人体内所整合HIV原病毒的相对定量。微滴式数字PCR(Droplet Digital PCR,ddPCR)技术可以用于分析极低拷贝DNA分子的定量,因此这里以ddPCR技术为代表来分析HIV原病毒基因组的相对含量。每个PCR孔使用700ng基因组DNA用于测定HIV原病毒的类型和/或相对含量,每个PCR孔使用7ng基因组DNA用于测定宿主基因组含量以便用于标定HIV原病毒的相对含量,每个PCR反应设置3个重复并采用健康人的CD4阳性T淋巴细胞基因组作为阴性对照、JLat6.3细胞基因组作为阳性对照。
2、依据病人特异性完整可感染性原病毒序列,设计相应特异性疫苗。
参照曾取得疗效的RV144疫苗,本发明基于原病毒序列的新型逆转录病毒疫苗也可以设计为基于prime/boost免疫策略的双组分系统:首先采用携带包含HIV-1Gag蛋白、Pro蛋白(protease)以及融合至gp41的gp120蛋白的金丝雀痘病毒载体(命名为ALVAC-HIVpro,pro为provirus缩写)感染待接种人员作为基础免疫(prime)(剂量:每株HIV-1均为106.5TCID50),然后采用gp120和铝佐剂的混合物(各300μg)疫苗(命名为AIDSpro,pro为provirus缩写)两次接种作为加强免疫(boost)。
3、疫苗使用。
疫苗于第0天(接种计划开始之日)、第4周(时间范围可以宽限为第3周到第7周)、第12周(时间范围可以宽限为第10周到第15周)、第24周(时间范围可以宽限为第21周到第28周)接种。ALVAC-HIVpro疫苗在4个时间点均要接种,加强免疫接种的AIDSpro疫苗则在第12周和第24周进行两次接种。每次接种后的第三天,受试者(包括疫苗接种者和安慰剂接种者)均要上报接种疫苗后局部和全身系统性疫苗反应并填写专用登记表格。无论何时出现不良反应乃至严重的副作用,计划实施人员均要认真记录并做好不良反应分级,以用于评估疫苗的副作用。具体评级参考美国国家过敏与感染性疾病研究院获得性免疫缺陷综合征分部提供的相应评级标准(http://rcc.tech-res.com/safetyandpharmacovigilance)。
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,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
序列表
<110> 张全志
<120> 逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaatctctag cagtggcgcc cgaacag 27
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgagggatct ctagttacca gagtc 25
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caggactcgg cttgctgaag 20
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcacccatct ctctccttct agc 23
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttttggcgta ctcaccagt 19
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agtggtgcag agagaaaaaa gagc 24
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtctggcctg taccgtcagc 20
<210> 8
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccttgggttc ttggga 16
<210> 9
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccttaggttc ttaggagc 18
<210> 10
<211> 9719
<212> DNA
<213> HIV-1 HXB2(HIV-1 HXB2)
<400> 10
tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180
acaaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatggaatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag 300
agctgcatcc ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacctgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgatgggaa 840
aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca 900
agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt 960
agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 1020
ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc 1080
aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaaaaa agcacagcaa 1140
gcagcagctg acacaggaca cagcaatcag gtcagccaaa attaccctat agtgcagaac 1200
atccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaatgc atgggtaaaa 1260
gtagtagaag agaaggcttt cagcccagaa gtgataccca tgttttcagc attatcagaa 1320
ggagccaccc cacaagattt aaacaccatg ctaaacacag tggggggaca tcaagcagcc 1380
atgcaaatgt taaaagagac catcaatgag gaagctgcag aatgggatag agtgcatcca 1440
gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatagca 1500
ggaactacta gtacccttca ggaacaaata ggatggatga caaataatcc acctatccca 1560
gtaggagaaa tttataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat 1620
agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta 1680
gaccggttct ataaaactct aagagccgag caagcttcac aggaggtaaa aaattggatg 1740
acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg 1800
ggaccagcgg ctacactaga agaaatgatg acagcatgtc agggagtagg aggacccggc 1860
cataaggcaa gagttttggc tgaagcaatg agccaagtaa caaattcagc taccataatg 1920
atgcagagag gcaattttag gaaccaaaga aagattgtta agtgtttcaa ttgtggcaaa 1980
gaagggcaca cagccagaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga 2040
aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc 2100
tggccttcct acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc 2160
ccaccagaag agagcttcag gtctggggta gagacaacaa ctccccctca gaagcaggag 2220
ccgatagaca aggaactgta tcctttaact tccctcaggt cactctttgg caacgacccc 2280
tcgtcacaat aaagataggg gggcaactaa aggaagctct attagataca ggagcagatg 2340
atacagtatt agaagaaatg agtttgccag gaagatggaa accaaaaatg atagggggaa 2400
ttggaggttt tatcaaagta agacagtatg atcagatact catagaaatc tgtggacata 2460
aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt 2520
tgactcagat tggttgcact ttaaattttc ccattagccc tattgagact gtaccagtaa 2580
aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa 2640
taaaagcatt agtagaaatt tgtacagaga tggaaaagga agggaaaatt tcaaaaattg 2700
ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat 2760
ggagaaaatt agtagatttc agagaactta ataagagaac tcaagacttc tgggaagttc 2820
aattaggaat accacatccc gcagggttaa aaaagaaaaa atcagtaaca gtactggatg 2880
tgggtgatgc atatttttca gttcccttag atgaagactt caggaagtat actgcattta 2940
ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac 3000
agggatggaa aggatcacca gcaatattcc aaagtagcat gacaaaaatc ttagagcctt 3060
ttagaaaaca aaatccagac atagttatct atcaatacat ggatgatttg tatgtaggat 3120
ctgacttaga aatagggcag catagaacaa aaatagagga gctgagacaa catctgttga 3180
ggtggggact taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg 3240
gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaagaca 3300
gctggactgt caatgacata cagaagttag tggggaaatt gaattgggca agtcagattt 3360
acccagggat taaagtaagg caattatgta aactccttag aggaaccaaa gcactaacag 3420
aagtaatacc actaacagaa gaagcagagc tagaactggc agaaaacaga gagattctaa 3480
aagaaccagt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga 3540
agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa 3600
caggaaaata tgcaagaatg aggggtgccc acactaatga tgtaaaacaa ttaacagagg 3660
cagtgcaaaa aataaccaca gaaagcatag taatatgggg aaagactcct aaatttaaac 3720
tgcccataca aaaggaaaca tgggaaacat ggtggacaga gtattggcaa gccacctgga 3780
ttcctgagtg ggagtttgtt aatacccctc ccttagtgaa attatggtac cagttagaga 3840
aagaacccat agtaggagca gaaaccttct atgtagatgg ggcagctaac agggagacta 3900
aattaggaaa agcaggatat gttactaata gaggaagaca aaaagttgtc accctaactg 3960
acacaacaaa tcagaagact gagttacaag caatttatct agctttgcag gattcgggat 4020
tagaagtaaa catagtaaca gactcacaat atgcattagg aatcattcaa gcacaaccag 4080
atcaaagtga atcagagtta gtcaatcaaa taatagagca gttaataaaa aaggaaaagg 4140
tctatctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagataaat 4200
tagtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagatg 4260
aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctgccacctg 4320
tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaagga gaagccatgc 4380
atggacaagt agactgtagt ccaggaatat ggcaactaga ttgtacacat ttagaaggaa 4440
aagttatcct ggtagcagtt catgtagcca gtggatatat agaagcagaa gttattccag 4500
cagaaacagg gcaggaaaca gcatattttc ttttaaaatt agcaggaaga tggccagtaa 4560
aaacaataca tactgacaat ggcagcaatt tcaccggtgc tacggttagg gccgcctgtt 4620
ggtgggcggg aatcaagcag gaatttggaa ttccctacaa tccccaaagt caaggagtag 4680
tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac 4740
atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 4800
ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 4860
aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 4920
gaaatccact ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa 4980
tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc attagggatt 5040
atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattagaaca 5100
tggaaaagtt tagtaaaaca ccatatgtat gtttcaggga aagctagggg atggttttat 5160
agacatcact atgaaagccc tcatccaaga ataagttcag aagtacacat cccactaggg 5220
gatgctagat tggtaataac aacatattgg ggtctgcata caggagaaag agactggcat 5280
ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 5340
gaactagcag accaactaat tcatctgtat tactttgact gtttttcaga ctctgctata 5400
agaaaggcct tattaggaca catagttagc cctaggtgtg aatatcaagc aggacataac 5460
aaggtaggat ctctacaata cttggcacta gcagcattaa taacaccaaa aaagataaag 5520
ccacctttgc ctagtgttac gaaactgaca gaggatagat ggaacaagcc ccagaagacc 5580
aagggccaca gagggagcca cacaatgaat ggacactaga gcttttagag gagcttaaga 5640
atgaagctgt tagacatttt cctaggattt ggctccatgg cttagggcaa catatctatg 5700
aaacttatgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc 5760
tgtttatcca ttttcagaat tgggtgtcga catagcagaa taggcgttac tcgacagagg 5820
agagcaagaa atggagccag tagatcctag actagagccc tggaagcatc caggaagtca 5880
gcctaaaact gcttgtacca attgctattg taaaaagtgt tgctttcatt gccaagtttg 5940
tttcataaca aaagccttag gcatctccta tggcaggaag aagcggagac agcgacgaag 6000
agctcatcag aacagtcaga ctcatcaagc ttctctatca aagcagtaag tagtacatgt 6060
aacgcaacct ataccaatag tagcaatagt agcattagta gtagcaataa taatagcaat 6120
agttgtgtgg tccatagtaa tcatagaata taggaaaata ttaagacaaa gaaaaataga 6180
caggttaatt gatagactaa tagaaagagc agaagacagt ggcaatgaga gtgaaggaga 6240
aatatcagca cttgtggaga tgggggtgga gatggggcac catgctcctt gggatgttga 6300
tgatctgtag tgctacagaa aaattgtggg tcacagtcta ttatggggta cctgtgtgga 6360
aggaagcaac caccactcta ttttgtgcat cagatgctaa agcatatgat acagaggtac 6420
ataatgtttg ggccacacat gcctgtgtac ccacagaccc caacccacaa gaagtagtat 6480
tggtaaatgt gacagaaaat tttaacatgt ggaaaaatga catggtagaa cagatgcatg 6540
aggatataat cagtttatgg gatcaaagcc taaagccatg tgtaaaatta accccactct 6600
gtgttagttt aaagtgcact gatttgaaga atgatactaa taccaatagt agtagcggga 6660
gaatgataat ggagaaagga gagataaaaa actgctcttt caatatcagc acaagcataa 6720
gaggtaaggt gcagaaagaa tatgcatttt tttataaact tgatataata ccaatagata 6780
atgatactac cagctataag ttgacaagtt gtaacacctc agtcattaca caggcctgtc 6840
caaaggtatc ctttgagcca attcccatac attattgtgc cccggctggt tttgcgattc 6900
taaaatgtaa taataagacg ttcaatggaa caggaccatg tacaaatgtc agcacagtac 6960
aatgtacaca tggaattagg ccagtagtat caactcaact gctgttaaat ggcagtctag 7020
cagaagaaga ggtagtaatt agatctgtca atttcacgga caatgctaaa accataatag 7080
tacagctgaa cacatctgta gaaattaatt gtacaagacc caacaacaat acaagaaaaa 7140
gaatccgtat ccagagagga ccagggagag catttgttac aataggaaaa ataggaaata 7200
tgagacaagc acattgtaac attagtagag caaaatggaa taacacttta aaacagatag 7260
ctagcaaatt aagagaacaa tttggaaata ataaaacaat aatctttaag caatcctcag 7320
gaggggaccc agaaattgta acgcacagtt ttaattgtgg aggggaattt ttctactgta 7380
attcaacaca actgtttaat agtacttggt ttaatagtac ttggagtact gaagggtcaa 7440
ataacactga aggaagtgac acaatcaccc tcccatgcag aataaaacaa attataaaca 7500
tgtggcagaa agtaggaaaa gcaatgtatg cccctcccat cagtggacaa attagatgtt 7560
catcaaatat tacagggctg ctattaacaa gagatggtgg taatagcaac aatgagtccg 7620
agatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat 7680
ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg 7740
tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag 7800
caggaagcac tatgggcgca gcctcaatga cgctgacggt acaggccaga caattattgt 7860
ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt 7920
tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat 7980
acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca 8040
ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca 8100
cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa 8160
ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat 8220
gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca 8280
taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga 8340
atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg 8400
gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca 8460
ttcgattagt gaacggatcc ttggcactta tctgggacga tctgcggagc ctgtgcctct 8520
tcagctacca ccgcttgaga gacttactct tgattgtaac gaggattgtg gaacttctgg 8580
gacgcagggg gtgggaagcc ctcaaatatt ggtggaatct cctacagtat tggagtcagg 8640
aactaaagaa tagtgctgtt agcttgctca atgccacagc catagcagta gctgagggga 8700
cagatagggt tatagaagta gtacaaggag cttgtagagc tattcgccac atacctagaa 8760
gaataagaca gggcttggaa aggattttgc tataagatgg gtggcaagtg gtcaaaaagt 8820
agtgtgattg gatggcctac tgtaagggaa agaatgagac gagctgagcc agcagcagat 8880
agggtgggag cagcatctcg agacctggaa aaacatggag caatcacaag tagcaataca 8940
gcagctacca atgctgcttg tgcctggcta gaagcacaag aggaggagga ggtgggtttt 9000
ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 9060
cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaaag aagacaagat 9120
atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattagca gaactacaca 9180
ccagggccag gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt 9240
gagccagata agatagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg 9300
agcctgcatg ggatggatga cccggagaga gaagtgttag agtggaggtt tgacagccgc 9360
ctagcatttc atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgacat 9420
cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg 9480
actggggagt ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg 9540
gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 9600
gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 9660
tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagca 9719
Claims (6)
1.一种逆转录病毒原病毒序列在逆转录病毒疫苗设计中的应用,其特征在于:针对特定个体或特定人群感染的原病毒序列,设计亚单位疫苗、灭活疫苗、减活疫苗、重组疫苗和核酸疫苗。
2.如权利要求1所述的应用,其特征在于:所述原病毒序列包括逆转录病毒全病毒序列或病毒某一个或某几个蛋白序列或蛋白结构域。
3.如权利要求1所述的应用,其特征在于:所述核酸疫苗包括DNA疫苗和RNA疫苗。
4.如权利要求1所述的应用,其特征在于:所述逆转录病毒包括RNA肿瘤病毒亚科、慢病毒亚科和泡沫病毒亚科家族中的所有成员。
5.如权利要求1所述的应用,其特征在于:所述逆转录病毒疫苗的接种途径包括皮内注射、皮下注射、肌内注射、口服。
6.如权利要求1所述的应用,其特征在于:所述逆转录病毒疫苗应用于人、猫、牛等物种逆转录病毒感染的非治疗目的应用和预防中。
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