CN107058353A - Hiv嵌合抗原、制备方法及应用 - Google Patents
Hiv嵌合抗原、制备方法及应用 Download PDFInfo
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- CN107058353A CN107058353A CN201710031642.1A CN201710031642A CN107058353A CN 107058353 A CN107058353 A CN 107058353A CN 201710031642 A CN201710031642 A CN 201710031642A CN 107058353 A CN107058353 A CN 107058353A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
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Landscapes
- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种HIV嵌合Gag抗原、制备方法及应用，具体公开了一种二价HIV嵌合Gag抗原，该抗原包含Gag嵌合序列1、Gag嵌合序列2及可溶性的PD1序列，同时还公开了抗原的制备方法及在预防、治疗HIV疾病中的应用及诊断HIV疾病的试剂中的应用。本发明二价HIV嵌合Gag抗原是基于中国数百株HIV‑1 B/B'，C/CRF07/08_BC和CRF01_AE Gag P41序列而设计的，其包含了最保守的T细胞抗原决定表位，本发明还在基于PD1的疫苗的研究成果上设计了对应的疫苗，该疫苗具有诱导广谱HIV特异性T细胞免疫的作用。
Description
技术领域
本发明涉及HIV抗原、制备方法及应用。具体而言，本发明涉及一种HIV嵌合Gag抗原、制备方法及应用。
背景技术
在过去的30年中，I型人类免疫缺陷病毒(HIV-1)是全球现有的最具破坏性的传染病微生物之一。至2014年，HIV-1已造成近78万人感染，其中一半死于获得性免疫缺陷综合症(AIDS)。尽管终生维持治疗能够延长HIV病人生命，但所产生的经济负担及耐药性毒株的出现给病人带来苦难。正是由于以上挑战，在过去三十年付出了巨大的努力去研发有效的HIV疫苗，然而在最近的泰国RV144临床试验中，也只有31％的保护率。另外，HIV-1的遗传多样性成为了全球疫苗发展的另一个挑战。理想情况下，一个有效的疫苗应该引起宿主的免疫反应以抑制和控制感染，但迄今为止，源于自然的HIV-1序列的抗原在非人灵长类动物的研究中和临床试验中只能引发有限度的细胞免疫应答。通过计算机算法设计嵌合抗原，能够匹配和最大化融合自然的HIV-1毒株序列。多价嵌合抗原是一种能够提高针对HIV-1多样性的细胞免疫覆盖率的优化免疫原，嵌合疫苗方法成为有潜力的解决方案。然而，针对中国流行的HIV-1B/B'、C/CRF07/08_BC和CRF 01_AE亚型的嵌合免疫原设计还没有报道。
发明内容
本发明要解决的技术问题是提供了了一种HIV嵌合Gag抗原、制备方法及应用。
本发明第一方面提供了一种核酸，所述核酸的序列选自：
1)包含SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列；或
2)核酸的同源序列，其与SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列有至少70％-99％的同一性；或
3)核酸的变体，其与SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列相比具有至少一个核苷酸的取代、缺失和/或添加；或
4)核酸的衍生物，其为1)-3)所述的核酸的序列的变体。
在一优选例中，所述的核酸进一步包含可溶性PD1序列。
在一优选例中，所述可溶性PD1序列为人类可溶性PD1序列。
在一优选例中，所述的人类可溶性PD1序列为SEQ ID NO:6所示的核苷酸序列。
在一优选例中，所述的核酸序列进一步包含作为链接体的linker序列。
在一优选例中，所述linker序列位于SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列之间。
在一优选例中，所述linker序列位于SEQ ID NO:6和SEQ ID NO:1所示的核苷酸序列之间。
在一优选例中，所述linker序列为SEQ ID NO:7所示的核苷酸序列。
在一优选例中，所述位于SEQ ID NO:6和SEQ ID NO:1所示的核苷酸序列之间的linker序列与SEQ ID NO:6之间含有第一酶切位点序列。
在一优选例中，所述第一酶切位点序列为EcoRI酶切序列。
在一优选例中，所述的核酸序列进一步包含第二酶切位点序列。
在一优选例中，所述第二酶切位点序列与SEQ ID NO:6相连。
在一优选例中，所述第二酶切位点序列为BamHI酶切序列。
在一优选例中，所述的核酸序列进一步包含组织纤溶酶原激活蛋白表达及释放的信号肽序列和起始密码子。
在一优选例中，所述BamHI酶切序列依次与组织纤溶酶原激活蛋白表达及释放的信号肽序列和起始密码子相连接。
在一优选例中，所述的核酸序列进一步包含第三酶切位点序列和第四酶切位点序列。
在一优选例中，所述SEQ ID NO:2所示的核苷酸序列依次与终止密码子、第三酶切位点序列和第四酶切位点序列相连接。
在一优选例中，所述的第三酶切位点序列和第四酶切位点序列分别为XhoI酶切序列和PmeI酶切序列。
在一优选例中，所述的核酸序列为SEQ ID NO:5序列。
本发明第二方面提供了一种氨基酸，由上述核酸序列编码。
本发明第三方面提供了一种载体，含有上述的核酸序列。
本发明第四方面提供了一种宿主细胞，其转化或转染了上述的载体。
本发明第五方面提供了一种疫苗，包含上述的核酸序列、或者上述的氨基酸序列、或者上述的载体、或者上述的宿主细胞。
所述疫苗还包含其他HIV疫苗。
所述包含的疫苗为基于PD1的疫苗；优选的还包含佐剂。
本发明第六方面提供了一种疫苗的制备方法，包含制备权利要求上述疫苗所需的步骤。
本发明第七方面提供了上述核酸序列、或者上述的氨基酸序列、或者上述的载体、或者上述的宿主细胞在制备预防和治疗HIV疾病的药物的应用。
本发明第八方面提供了上述核酸序列、或者上述的氨基酸序列、或者上述的载体、或者上述的宿主细胞在制备HIV疾病诊断试剂中的应用。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子生物学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，EpitopeMapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得扩增和/或表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
如本文中所使用的，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-ASynthesis(第2版，E.S.Golub和D.R.Gren，Eds.，Sinauer Associates，Sunderland，Mass.(1991))，其通过引用合并入本文。
本发明具体公开了一种二价HIV嵌合Gag抗原，该抗原包含Gag嵌合序列1、Gag嵌合序列2及可溶性的PD1序列，同时还公开了抗原的制备方法及在预防、治疗HIV疾病中的应用及诊断HIV疾病的试剂中的应用。本发明二价HIV嵌合Gag抗原是基于中国数百株HIV-1B/B'，C/CRF07/08_BC和CRF01_AE Gag P41序列而设计的，其包含了最保守的T细胞抗原决定表位，本发明还在基于PD1的疫苗的研究成果上设计了对应的疫苗，该疫苗具有诱导广谱HIV特异性T细胞免疫的作用。
附图说明
图1：编码二价HIV嵌合Gag抗原的Gag p41mosaic1和Gag p41mosaic2的核酸序列。
图2：与图1的核酸序列对应的氨基酸序列。
图3：两个HIV-1Gag嵌合抗原氨基酸序列T细胞抗原决定表位覆盖率分析图。
图4:基于PD1的二价HIV嵌合Gag抗原结构图。
图5:Western blot图：二价HIV嵌合Gag抗原的表达和从293T细胞中的释放。
图6:本发明二价HIV嵌合Gag抗原(mosaic)的疫苗在小鼠模型中诱导出针对中国三种亚型(HIV-1B/B'，C/CRF07/08_BC和CRF01_AE)的T细胞反应的ELISPOT结果图。图中横坐标为检测项目，包括B亚型多肽、BC亚型多肽和AE亚型多肽，纵坐标为每百万脾细胞ELISPOT数。右方标注：PBS指代阴性对照，p24Fc是pVAX-P24Fc的简写，msPD1-p24Fc是pVAX-msPD1-p24Fc的简写，mosaic是pVAX-mosaic的简写，huPD1-mosaic是pVAX-huPD1-mosaic的简写。
图7:本发明二价HIV嵌合Gag抗原的疫苗在恒河猴模型中诱导出T细胞反应的ELISPOT结果图。图7A示出猴疫苗免疫试验的时间安排，横轴上的数字代表免疫时间/取样时间；图7B示出二十八周间，不同时间点，四只猴针对B亚型P24蛋白的三个不交叉肽库的T细胞反应；图7C示出二十八周间，四只猴针对三个亚型P17和P24蛋白的四个不交叉肽库的T细胞反应；图7D示出第二十八周间，四只猴针对AE亚型P17和P24蛋白的单一肽的T细胞反应，数字是单一肽的编号。
图8:用二价HIV嵌合Gag抗原的肽库分为Mosaic p41-1(上)和Mosaic p41-2(下)检测中国自然感染HIV病人的T细胞反应的ELISPOT结果图。
具体实施方式
除非特殊说明，本发明所用术语具有本发明所属领域中的一般含义。
下面参考具体实施例和附图，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，均按照常规实验条件，或按照制造厂商说明书建议的条件。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：二价HIV嵌合Gag抗原设计
1.抗原序列选择：
理想情况下，HIV-1的变种和分支之间的共同保守的抗原表位是常见的，因此可以进一步加强对多样性的自然株的防护。本实施例通过同源性抗原表位分析(http://www.hiv.lanl.gov/content/sequence/MOSAIC/)，设计了一个基于数百种中国流行的HIV-1B/B'，C/CRF07/08_BC和CRF01_AE亚型的病毒株序列，包括最常见的保守表位的二价HIV嵌合Gag抗原。
本实施例二价HIV嵌合Gag抗原的设计选择了二价HIV Gag p41基因的嵌合DNA与人类PD-1(也称为PDCD1或CD279)的可溶性结构域融合，其目的是为了提高疫苗的反应性。所述人类PD-1的可溶性结构域代表基因密码子优化后的可溶性的PD1组分，即基因密码子优化后的原始PD1表达在细胞外的部分，不含转膜和细胞内的部分，基因密码子优化的目的是增强蛋白在人细胞内的表达量。
所选的二价HIV Gag p41基因的嵌合DNA包括编码嵌合1抗原的核酸序列(Gagp41mosaic1)和编码嵌合2抗原的核酸序列(Gag p41mosaic 2)。
编码嵌合1抗原的核酸序列为:
ATGGGGGCAAGAGCCTCCGTGCTGTCTGGCGGGAAACTGGACGCCTGGGAGAAGATCCGGCTGAGACCAGGAGGCAAGAAAAAGTACCGCCTGAAGCACATCGTGTGGGCATCCCGCGAACTGGAGCGATTCGCCCTGAACCCAGGACTGCTGGAAACCGCAGAGGGATGCCAGCAGATCATTGAGCAGCTGCAGTCTACACTGAAAACTGGCTCCGAGGAACTGAAGTCTCTGTTTAACACCATCGCTGTGCTGTGGTGCGTGCATCAGCGCATTGACGTGAAGGATACAAAAGAGGCCCTGGACAAGATCGAGGAAGTGCAGAACAAGTCACAGCAGAAGACTCAGCAGGCCGCTGCAGGAACCGGAAGCTCCTCTAAGGTGAGCCAGAACTATCCCATTGTCCAGAATGCACAGGGACAGATGGTGCACCAGCCACTGAGCCCTCGGACCCTGAACGCATGGGTGAAAGTGGTCGAGGAAAAGGGCTTCAATCCTGAAGTCATCCCAATGTTTAGTGCACTGTCAGAGGGGGCCACACCTCAGGATCTGAACATGATGCTGAATATCGTGGGGGGACATCAGGCCGCTATGCAGATGCTGAAGGAAACTATTAATGAGGAAGCAGCAGAGTGGGACCGAGTGCACCCAGTCCATGCAGGACCAATCCCACCTGGACAGATTCGAGAACCACGAGGATCCGATATCGCCGGCACCACATCTACTCTGCAGGAGCAGATTGGGTGGATGACCAACAATCCACCCATCCCTGTGGGAGACATCTACAAACGCTGGATCATTCTGGGCCTGAACAAGATCGTGCGAATGTATAGCCCAGTCTCCATCCTGGATATTCGGCAGGGACCAAAAGAGCCCTTCAGGGACTACGTGGATCGCTTTTATAAGACACTGAGAGCAGAACAGGCCACTCAGGAGGTGAAAAATTGGATGACAGAGACTCTGCTGGTCCAGAACGCCAATCCTGACTGCAAATCTATTCTGAAGGCTCTGGGGACCGGAGCAACACTGGAGGAAATGATGACCGCTTGTCAGGGAGTGGGAGGACCAGGACACAAGGCAAGGGTCCTG(SEQ ID NO:1)
编码嵌合2抗原的核酸序列为：
ATGGGCGCCCGAGCCAGCATCCTGCGGGGAGGCAAGCTGGATAAATGGGAGAAGATTAGGCTGCGCCCCGGGGGAAAAAAGCACTACATGCTGAAGCATCTGGTGTGGGCTTCTCGGGAACTGGAGAGATTCGCAGTCAACCCAGGCCTGCTGGAAACCAGTGAGGGGTGCAAACAGATCATTAAGCAGCTGCAGCCCGCTCTGCAGACCGGAACAGAGGAACTGCGCAGTCTGTTTAACACTGTGGCCACCCTGTACTGCGTGCACCAGCGAATCGAGATCAAGGACACAAAGGAGGCCCTGGATAAAATCGAGGAAGAGCAGAATAAGTCCAAAAAGAAAGCTCAGCAGACAGCTGCAGATACTGGAAACAATTCTCAGGTGAGTCAGAACTATCCAATCGTCCAGAATCTGCAGGGCCAGATGGTGCACCAGCCTATTAGCCCAAGAACCCTGAACGCCTGGGTGAAAGTGGTCGAAGAGAAGGCTTTCAGCCCCGAAGTCATCCCTATGTTTACCGCCCTGTCCGAGGGAGCTACACCTCAGGACCTGAACACCATGCTGAATACAGTGGGCGGGCACCAGGCTGCTATGCAGATCCTGAAGGACACTATTAATGAAGAGGCAGCCGAGTGGGATAGGCTGCACCCAGTGCATGCAGGACCAGTCGCTCCTGGACAGATGAGAGAACCTAGGGGAAGTGATATCGCCGGCACTACCTCAAACCTGCAGGAGCAGATTGGCTGGATGACAAGCAATCCTCCAATCCCCGTGGGGGAAATCTACAAAAGATGGATCATTCTGGGACTGAACAAGATCGTGAGGATGTATTCACCTACTAGCATCCTGGACATCAAGCAGGGGCCAAAGGAGCCCTTCAGAGACTATGTGGATAGGTTCTTTAAGACCCTGAGAGCTGAACAGGCATCCCAGGACGTGAAAAATTGGATGACTGATACCCTGCTGGTCCAGAACGCAAATCCTGATTGCAAAACAATCCTGAAGGCCCTGGGCCCAGCTGCAACTCTGGAGGAGATGATGACCGCTTGCCAGGGCGTGGGAGGACCTTCACATAAAGCCAGAGTGCTG(SEQ ID NO:2)
编码嵌合1抗原的核酸序列所对应的氨基酸序列为：
MGARASVLSGGKLDAWEKIRLRPGGKKKYRLKHIVWASRELERFALNPGLLETAEGCQQIIEQLQSTLKTGSEELKSLFNTIAVLWCVHQRIDVKDTKEALDKIEEVQNKSQQKTQQAAAGTGSSSKVSQNYPIVQNAQGQMVHQPLSPRTLNAWVKVVEEKGFNPEVIPMFSALSEGATPQDLNMMLNIVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIPPGQIREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKSILKALGTGATLEEMMTACQGVGGPGHKARVL((SEQ ID NO:3)
编码嵌合2抗原的核酸序列所对应的氨基酸序列为：
MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFAVNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQTAADTGNNSQVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVVEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQILKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIGWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQASQDVKNWMTDTLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPSHKARVL(SEQ ID NO:4)
编码嵌合1抗原的核酸序列和编码嵌合2抗原的核酸序列对比结果如图1所示。
编码嵌合1抗原的核酸序列所对应的氨基酸序列和编码嵌合2抗原的核酸序列所对应的氨基酸序列对比结果如图2所示。
2.覆盖率分析：
针对中国流行的HIV-1 B/B'(176株)、HIV-1 01_AE(236株)和HIV-1 C /BC(127株)亚型的序列(来自Genbank，对应ID包括AB078678-AB078687；AB078689-AB078690；AB078692-AB078704；AB078709；AB078711；AB213667-AB213689；AB213692；AB746342；AF286226；AF286229；AF286230；AF503396；AY008714；AY008716；AY180905；AY275555-AY275557；DD033495；DQ833405-DQ833436；DQ859178-DQ859180；EF036527-EF036536；EF122502-EF122505；EF122507-EF122511；EF122513-EF122522；EF122524-EF122526；EF122528-EF122545；EF122559；EF368370-EF368372；EF394231-EF394236；EF420986；FJ441290-FJ531390；FJ531405-FJ531445；GQ845124-GQ845126；GU177863；GU564221-GU564225；GU564227-GU564230；HM067748；HQ197984-HQ197989；HQ215552-HQ215556；HQ215568-HQ215577；JF719819；JF932468-JF932500；JQ028170；JQ028172-JQ028173；JQ028179；JQ028187-JQ028188；JQ028194-JQ028195；JQ028208-JQ028211；JQ028213-JQ028214；JQ028217；JQ028220-JQ028221；JQ028223；JQ028228-JQ028231；JQ028236；JQ028239；JQ028241-JQ028242；JQ028244；JQ028246-JQ028256；JQ028271；JQ028285；JQ028288-JQ028289；JQ028291；JQ028293；JQ028295；JQ028299；JQ028617-JQ028618；JQ028624；JQ028633；JQ028638；JQ028646-JQ028649；JQ028653-JQ028661；JQ234979-JQ235007；JQ423923；JQ898186；JQ898208；JQ898220；JQ900844-JQ900942；JX140658；JX392347-JX392362；JX392378-JX392384；JX960597-JX960634；KC596061-KC596066；U71182.)，分析编码嵌合1抗原的核酸序列所对应的氨基酸序列和编码嵌合2抗原的核酸序列所对应的氨基酸序列(即SEQ ID NO:3和SEQ ID NO:4)的T细胞抗原决定表位覆盖率，分析结果如图3所示(图3中的AE对应HIV-1 CRF01_AE，B对应HIV-1 B/B'，BC对应HIV-1 C,CRF07_BC和CRF08_BC)。
结果显示，如果以总体和每个亚型的T细胞九肽表位中九个氨基酸序列完全一致(Exact match，9/9match)计算，覆盖率均达到70％左右；如果以T细胞九肽表位中有八个氨基酸序列完全一致(8/9match)计算，覆盖率均达到90％左右；如果以T细胞九肽表位中有七个氨基酸序列完全一致(7/9match)计算，覆盖率均达到95％左右。其中，又以对中国性传播为主的HIV-101_AE亚型病毒的覆盖率最高。
实施例2：基于PD1的二价HIV嵌合Gag抗原的构建
二价HIV嵌合Gag抗原结构如图4所示，其中CMV代表CMV启动子，tPA代表组织纤溶酶原激活蛋白表达及释放的信号肽序列，sPD1代表可溶性的PD1组分，为对应的核苷酸或氨基酸序列只包含PD1蛋白位于胞外区的序列，每个linker(链接体)含(G4S)3氨基酸序列，(G4S)3氨基酸序列代表三个GGGGS的重复氨基酸序列。
基于PD1的二价HIV嵌合Gag抗原更具体的序列组成如下：
START-tPA-BamHI-opt-huPD1-EcoRI-linker-Gag p41mosaic1-linker-Gagp41mosaic 2-STOP-XhoI-PmeI
其中START代表基因起始密码子ATG，tPA代表组织纤溶酶原激活蛋白表达及释放的信号肽序列,BamHI、EcoRI、XhoI和PmeI代表对应的限制性内切酶酶切位点，opt-huPD1代表基因密码子优化后的可溶性的PD1组分，即基因密码子优化后的原始PD1表达在细胞外的部分，不含跨膜区和细胞内的部分，STOP代表基因终止密码子，每个linker(链接体)含(G4S)3氨基酸序列，(G4S)3代表三个GGGGS的重复氨基酸序列。
START-tPA-BamHI-opt-huPD1-EcoRI-linker-Gag p41mosaic1-linker-Gagp41mosaic 2-STOP-XhoI-PmeI对应的具体核酸序列如下：
ATGGACGCCATGCTGCGCGGACTGTGCTGCGTGCTGCTACTGTGCGGCGCCGTGTTCGTGAGCCCCAGCCAGGAGATCCACGCCCGATTCAGGAGAGGAGCCAGAGGAGGATCCATGCAGATTCCTCAGGCTCCATGGCCTGTGGTGTGGGCAGTGCTGCAGCTGGGATGGAGACCAGGATGGTTCCTGGACTCCCCTGACAGACCATGGAATCCCCCTACATTTTCTCCTGCACTGCTGGTGGTGACTGAGGGCGATAACGCCACCTTCACATGCAGCTTTTCCAACACTTCTGAAAGTTTCGTCCTGAATTGGTACAGGATGTCACCCAGCAACCAGACTGACAAGCTGGCCGCTTTTCCCGAAGACCGCTCCCAGCCTGGGCAAGATTGCCGATTCCGGGTGACACAGCTGCCTAATGGAAGGGACTTTCACATGAGTGTGGTCCGCGCTCGGAGAAACGATTCAGGAACCTATCTGTGTGGCGCAATCAGCCTGGCCCCTAAGACACAGATCAAGGAGAGCCTGAGAGCCGAACTGAGGGTGACTGAGAGGCGCGCTGAAGTCCCAACCGCACATCCTTCCCCATCTCCCCGACCAGCAGGACAGGAATTCCGGGGAGGCGGGGGAAGTGGAGGAGGAGGATCCGGAGGAGGAGGAAGCATGGGGGCAAGAGCCTCCGTGCTGTCTGGCGGGAAACTGGACGCCTGGGAGAAGATCCGGCTGAGACCAGGAGGCAAGAAAAAGTACCGCCTGAAGCACATCGTGTGGGCATCCCGCGAACTGGAGCGATTCGCCCTGAACCCAGGACTGCTGGAAACCGCAGAGGGATGCCAGCAGATCATTGAGCAGCTGCAGTCTACACTGAAAACTGGCTCCGAGGAACTGAAGTCTCTGTTTAACACCATCGCTGTGCTGTGGTGCGTGCATCAGCGCATTGACGTGAAGGATACAAAAGAGGCCCTGGACAAGATCGAGGAAGTGCAGAACAAGTCACAGCAGAAGACTCAGCAGGCCGCTGCAGGAACCGGAAGCTCCTCTAAGGTGAGCCAGAACTATCCCATTGTCCAGAATGCACAGGGACAGATGGTGCACCAGCCACTGAGCCCTCGGACCCTGAACGCATGGGTGAAAGTGGTCGAGGAAAAGGGCTTCAATCCTGAAGTCATCCCAATGTTTAGTGCACTGTCAGAGGGGGCCACACCTCAGGATCTGAACATGATGCTGAATATCGTGGGGGGACATCAGGCCGCTATGCAGATGCTGAAGGAAACTATTAATGAGGAAGCAGCAGAGTGGGACCGAGTGCACCCAGTCCATGCAGGACCAATCCCACCTGGACAGATTCGAGAACCACGAGGATCCGATATCGCCGGCACCACATCTACTCTGCAGGAGCAGATTGGGTGGATGACCAACAATCCACCCATCCCTGTGGGAGACATCTACAAACGCTGGATCATTCTGGGCCTGAACAAGATCGTGCGAATGTATAGCCCAGTCTCCATCCTGGATATTCGGCAGGGACCAAAAGAGCCCTTCAGGGACTACGTGGATCGCTTTTATAAGACACTGAGAGCAGAACAGGCCACTCAGGAGGTGAAAAATTGGATGACAGAGACTCTGCTGGTCCAGAACGCCAATCCTGACTGCAAATCTATTCTGAAGGCTCTGGGGACCGGAGCAACACTGGAGGAAATGATGACCGCTTGTCAGGGAGTGGGAGGACCAGGACACAAGGCAAGGGTCCTGGGAGGCGGGGGAAGTGGAG GAGGAGGATCCGGAGGAGGAGGAAGCATGGGCGCCCGAGCCAGCATCCTGCGGGGAGGCAAGCTGGATAAATGGGAGAAGATTAGGCTGCGCCCCGGGGGAAAAAAGCACTACATGCTGAAGCATCTGGTGTGGGCTTCTCGGGAACTGGAGAGATTCGCAGTCAACCCAGGCCTGCTGGAAACCAGTGAGGGGTGCAAACAGATCATTAAGCAGCTGCAGCCCGCTCTGCAGACCGGAACAGAGGAACTGCGCAGTCTGTTTAACACTGTGGCCACCCTGTACTGCGTGCACCAGCGAATCGAGATCAAGGACACAAAGGAGGCCCTGGATAAAATCGAGGAAGAGCAGAATAAGTCCAAAAAGAAAGCTCAGCAGACAGCTGCAGATACTGGAAACAATTCTCAGGTGAGTCAGAACTATCCAATCGTCCAGAATCTGCAGGGCCAGATGGTGCACCAGCCTATTAGCCCAAGAACCCTGAACGCCTGGGTGAAAGTGGTCGAAGAGAAGGCTTTCAGCCCCGAAGTCATCCCTATGTTTACCGCCCTGTCCGAGGGAGCTACACCTCAGGACCTGAACACCATGCTGAATACAGTGGGCGGGCACCAGGCTGCTATGCAGATCCTGAAGGACACTATTAATGAAGAGGCAGCCGAGTGGGATAGGCTGCACCCAGTGCATGCAGGACCAGTCGCTCCTGGACAGATGAGAGAACCTAGGGGAAGTGATATCGCCGGCACTACCTCAAACCTGCAGGAGCAGATTGGCTGGATGACAAGCAATCCTCCAATCCCCGTGGGGGAAATCTACAAAAGATGGATCATTCTGGGACTGAACAAGATCGTGAGGATGTATTCACCTACTAGCATCCTGGACATCAAGCAGGGGCCAAAGGAGCCCTTCAGAGACTATGTGGATAGGTTCTTTAAGACCCTGAGAGCTGAACAGGCATCCCAGGACGTGAAAAATTGGATGACTGATACCCTGCTGGTCCAGAACGCAAATCCTGATTGCAAAACAATCCTGAAGGCCCTGGGCCCAGCTGCAACTCTGGAGGAGATGATGACCGCTTGCCAGGGCGTGGGAGGACCTTCACATAAAGCCAGAGTGCTGTGATAACCGCTCGAGCGGCCGGCGCGCCGTTTAAACAAAGCT(SEQ ID NO:5)
其中人可溶性的PD1基因密码子优化后的核酸(opt-huPD1)序列为：
ATGCAGATTCCTCAGGCTCCATGGCCTGTGGTGTGGGCAGTGCTGCAGCTGGGATGGAGACCAGGATGGTTCCTGGACTCCCCTGACAGACCATGGAATCCCCCTACATTTTCTCCTGCACTGCTGGTGGTGACTGAGGGCGATAACGCCACCTTCACATGCAGCTTTTCCAACACTTCTGAAAGTTTCGTCCTGAATTGGTACAGGATGTCACCCAGCAACCAGACTGACAAGCTGGCCGCTTTTCCCGAAGACCGCTCCCAGCCTGGGCAAGATTGCCGATTCCGGGTGACACAGCTGCCTAATGGAAGGGACTTTCACATGAGTGTGGTCCGCGCTCGGAGAAACGATTCAGGAACCTATCTGTGTGGCGCAATCAGCCTGGCCCCTAAGACACAGATCAAGGAGAGCCTGAGAGCCGAACTGAGGGTGACTGAGAGGCGCGCTGAAGTCCCAACCGCACATCCTTCCCCATCTCCCCGACCAGCAGGACAG(SEQ ID NO:6)
实施例3：二价HIV嵌合Gag抗原表达pVAX1表达质粒的构建
本实施例二价HIV嵌合Gag抗原表达pVAX1表达质粒的构建包括如下步骤：
(1)制备PCR扩增产物：经过分析539株HIV的序列(即上述提及的HIV-1B/B'(176株)、HIV-1CRF01_AE(236株)和HIV-1C/CRF07/08_BC(127株)亚型的序列)，取保守序列，通过体外合成上述编码嵌合1抗原的核酸序列(SEQ ID NO:1)-linker-编码嵌合2抗原的核酸序列(SEQ ID NO:2)，将合成的序列稀释到10uM浓度，取5ul为模板进行PCR扩增，PCR反应体系(PrimeSTAR HS DNA Polymerase,Takara货号R010A):共25ul反应体系,包括5ul模板，5μl的5x缓冲液,4μl的2.5mM dNTP,各1μl的20pM正向引物和反向引物,1μl的Taq酶(2.5units/μl),3μl的水。PCR反应条件:92℃2分钟；[92℃10秒；55℃15秒；68℃1.5分钟]33周期；68℃7分钟；4℃5分钟。所述正向引物(p41-1with linker)的序列为:5’-CCGGAATTCCGGGGAGGC GGGGGAAGT GGAGGAGGA GGATCCGGA GGAGGAGGA AGCATGGGG GCAAGAGCC TCC-3’(SEQ ID NO:8)；所述反向引物(p41-2)的序列为：5’-CCGGCGCGC CGTTTAAAC AAAGCTCCGCTCGAGCGG TTATCACAG CACTCTGGC TTTATG-3’(SEQ ID NO:9)。通过上述反应体系和反应条件进行PCR扩增反应即得到PCR扩增产物。
(2)制备pVAX1-huPD1表达载体：先合成上述人可溶性的PD1基因密码子优化后的核酸(opt-huPD1，即SEQ ID NO:6)，然后将opt-huPD1通过核酸连接酶，接入表达质粒载体pVAX1(Invitrogen,Carlsbad,CA货号V26020)中，形成含有序列优化的人可溶性pVAX1-huPD1表达载体。
(3)制备pVAX-huPD1-mosaic表达质粒：然后再使用EcoR1/XhoI(New EnglandBiosystems)双酶切含有序列优化的人可溶性pVAX1-huPD1表达载体,以切掉多余片段，然后利用其酶切位点连接同样经此双酶切的上述PCR扩增产物，连接后的产物再转染DH5alpha感受态菌(GenScript,Cat.No.M00086)，以上各操作均可按照对应的试剂说明书或现有常规方法进行，由此制备了pVAX-huPD1-mosaic的表达质粒。
接着对应制备了不含可溶性PD1的表达质粒pVAX-mosaic做为对照(即按照上述步骤但不与opt-huPD1连接)，以及按照上述步骤还制备了相应的小鼠可溶性PD1(GenBank#NM_008798)表达质粒pVAX-msPD1-mosaic和恒河猴可溶性PD1(GenBank#NM_001114358)pVAX-rhPD1-mosaic。
将上述构建的四种pVAX1表达质粒通过聚乙烯亚胺(PEI，polysciences,Inc.Cat:23966)转染293T细胞(ATCC CRL-1573TM，下同)，获得表达上清并进行Western blot检测。
具体步骤为：1)接种细胞
转染前24小时，24孔板每孔接种0.5-2×105个293T细胞(ATCC货号CRL-11268),使其转染密度为60-80％。每孔接种细胞分别培养在0.5ml-1ml的培养基(DMEM(LifeScience,货号111995073)中。该培养基含有10％胎牛血清(FBS，Life science,货号110270106)和1％抗生素(Life science,货号1 15140122)。
2)制备转染复合物，细胞转染
制备转染复合物：分别将上述构建的四种pVAX1表达质粒各1μg稀释于50μl optiMEM液(Life science，货号31985070，下同)，轻轻混匀，得到50μl DNA稀释液；4μl PEI(1mg/ml，polysciences,Inc.货号23966)稀释于50μl opti MEM液，轻轻混匀，然后将当中的50μl PEI稀释液取出；再滴加到50μl DNA稀释液中，轻轻混匀,室温孵育10-15分钟，获得100μl PEI/DNA复合物。
细胞转染：将上述每孔接种细胞的上述含抗生素的培养基更换为新鲜的不含抗生素的培养基，该培养基含有10％胎牛血清(Life science,货号110270106)。然后，将100μlPEI/DNA复合物缓慢地加到每孔中并轻轻摇动使其均匀混合，然后放置于37℃的CO2孵育箱孵育6-8小时后，更换为新鲜的含有1％抗生素的培养基。
3)Western blot检测
在细胞中加入复合物转染72小时后，分别收集转染细胞培养液经Western blot检测表达产物并验证表达产物是否释放。
转染细胞培养液用样品稀释液(样品稀释液内含：50mM of Tris-HCl[pH 8.0]；137mM of NaCl；2mM of EDTA；0.5％NP-40裂解液；10％甘油；1mg/mL的胃蛋白酶抑制剂(pepstatin),1mg/mL亮肽素(leupeptin),1mg/mL胰蛋白酶抑制剂(pefabloc)稀释(转染细胞培养液培养液与样品稀释液体积比是1:5)，然后在95℃下加热10分钟，然后将其加到浓度为10％的丙烯酰胺凝胶中，电泳条件设定在80伏电压30分钟，然后上调到130伏电压1个小时，电泳结束后蛋白质T条带半干转转膜到PVDF膜上(13伏电压小于1小时，Bio-Rad的Semi-Dry Transfer Cell Trans-Blot SD)。PVDF膜用PBS洗三次，然后在封闭液(即PBS含有5％脱脂奶粉和0.5％牛血清白蛋白，下同)中室温1小时或者4℃过夜。一抗(Anti-HIV-1p24Hybridoma(183-H12-5C)(NIH货号1513))用封闭液稀释(体积比是1:5000)后加到PVDF膜，室温反应2小时，然后用PBS充分洗涤。二抗(荧光标记的抗小鼠、兔和人的抗体Lico,货号P/N 926-68078)用封闭液稀释(体积比是1:10000)后继续加到PVDF膜，室温反应1小时，PVDF膜用PBS洗涤后自然干燥，接着用LI-COR扫描仪进行蛋白条带扫描。试验包括了四种疫苗抗原的表达比较：mosaic，小鼠msPD1-mosaic，人huPD1-mosaic和猴rhPD1-mosaic，mosaic即pVAX-mosaic上清。
结果如图5所示，从图5中可知，二价HIV嵌合Gag抗原(mosaic)本身不能有效释放，但二价HIV嵌合Gag抗原与小鼠(msPD1-mosaic)、人(huPD1-mosaic)和猴子(rhPD1-mosaic)可溶性PD1的融合蛋白都能有效表达并释放到了细胞外。
实施例4：小鼠实验
本实施例是将Balb/c小鼠经肌肉电转100ug DNA疫苗后，ELISPOT用于检测Gagp17和Gag p24刺激的脾脏CD4+和CD8+T淋巴细胞分泌IFN-y的能力。本实施例是在香港大学实验动物中心的标准无菌条件的饲养间里进行，此使用活体动物实验是经由大学委员会批准。活体动物采用六到八周龄的雌性BALB/c小鼠，其日常护理为：将六到八周龄的雌性BALB/c小鼠饲养在常规标准温度和湿度的下的笼子里，食物和水自由采食，由香港大学实验动物中心的专职人员进行日常照管。
具体方案如下：
(1)免疫处理
通过肌肉注射，每组小鼠分别接受三次100微升的pVAX-huPD1-mosaic(即实施例3制备得到的pVAX-huPD1-mosaic的表达质粒)、pVAX-mosaic(即实施例3中的作为对照的表达质粒pVAX-mosaic)以及已经报道过的pVAX-P24Fc和pVAX-msPD1-p24Fc(Zhou，etal.PD1-based DNA vaccine amplifies HIV-1GAG-specific CD8+T cells inmice.Journal of Clinical Investigation 2013,123:2629-2642.)的电击(EP60V)免疫注射。免疫注射3次的时间分别为第0周、第3周和第6周，每只小鼠3次免疫剂量分别为4μg、20ug、100ug(即实际DNA含量)。每一次免疫后两周，采集血液样本进行血清检测。最后一次免疫后的两周(标准短期)或30周(长期)，处死小鼠，收集或制备血浆、脾脏和外周血单个核细胞，进行免疫反应分析。
(2)获得目标细胞
处死小鼠的脾脏用3ml淋巴细胞分离缓冲液(达科为生物技术有限公司，货号DKW33-R0100)进行分离，然后将分离的细胞通过70微米(μm)过滤器(BD公司)获得单细胞。小心将单细胞用1ml RPMI1640培养液(Life Technologies Limited,货号21870092)悬起，转移到15ml离心管中进行800g 30分钟无制动离心，仔细收集中间层，然后用RPMI1640培养液洗涤三次，得到目标细胞。
(3)HIV-1gag特异性T细胞反应的评估
将步骤(2)得到的目标细胞用100μL RPMI1640培养液稀释到适当的浓度(通常每孔2×105细胞/100微升)，并同时加入每种肽终浓度均为2μg/ml的HIV-1gag(p17和p24)B亚型多肽、C亚型多肽(均由美国国立卫生研究院提供，目录依次为8117号和8118号)和AE亚型多肽(均由吉尔生化上海有限公司合成)，进行脾细胞体外刺激。
阳性对照：区别在于，目标细胞用500ng/ml的佛波醇12-mystrate 13-acetate(PMA；Sigma-Aldrich)加上1μg/ml钙离子霉素刺激。
阴性对照：区别在于，目标细胞不加肽刺激物，只含细胞培养液(即RPMI1640培养液)。
分别将前述的加入多肽的目标细胞、阳性对照和阴性对照的细胞在37℃，5％CO2，100％湿度条件下孵育20小时，再用ELISPOT检测(采用Diaclone的Murine IFN-γELISpotkit试剂盒，具体步骤按照试剂盒的操作方法进行)脾脏CD4+和CD8+T淋巴细胞分泌IFN-y的能力，将细胞形成的斑点显色后用免疫斑点测试仪扫描再用图像分析仪(ThermoScientific)进行实验数据分析。
图6中三个病毒亚型肽库的来源于：HIV Consensus B Gag peptides Set(Cat#8117,Lot#10080196,NIH AIDS Reagent Program),HIV Consensus C Gag peptides(Cat#8118,Lot#3,NIH AIDS Reagent Program),以及HIV Consensus 01_AE Gag peptides(按照01_AE Gag的完整序列，来源于中国感染者，通过分析确定为如下氨基酸序列：
HIV 01_AE Gag P17(SEQ ID NO:12):
MGARASVLSGGKLDAWEKIRLRPGGKKKYRMKHLVWASRELERFALNPGLLETAEGCQQGCQQLQSTLKTGSEELKSLFNTVATLWCVHQRIDVKDTKEALDKIEEVQNKSQQKTQQAAAGTGSSSKVSQNYPIV；
以及HIV 01_AE Gag P24(SEQ ID NO:13):
QNAQGQMVHQPVSPRTLNAWVKVVEEKGFNPEVIPMFSALSEGATPQDLNMMLNIVGGHQAAMQMLKETINEEAADWDRTWDRTAGPIPPGQIREPRGSDIAGTTSTLQEQIAWMTNNPPIPVGDIYKRWIILGLNKIVRMYSPPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKSILKALGTGATLEEMMTACQGVGGPSHKARVL。从头到尾依次合成15个氨基酸的肽，下一个与前一个有11个氨基酸的重叠，即15-meroverlap by 11的常规方法)。在BALB/c小鼠中，B亚型P24的单肽GAG[GHAQAAMQMLKETINE](SEQ ID NO:14)，是针对特定的小鼠CD8+T细胞，而B亚型P24单肽GAG[TNNPPIPVGEIYKRWIILGLN](SEQ ID NO:15)，是针对特定的小鼠CD4+T细胞；C/BC亚型P24的单肽GAG[GGHQAAMQMLKDTIN](SEQ ID NO:16)，是针对特定的小鼠CD8+T细胞，而C/BC亚型P24单肽GAG[TSNPPVPVGDIYKRWIILGL](SEQ ID NO:17)，是针对特定的小鼠CD4+T细胞；AE亚型P24的单肽GAG[GHQAAMQMLKETINE](SEQ ID NO:18)是针对特定的小鼠CD8+T细胞，而AE亚型P24单肽GAG[TNNPPIPVGDIYKRWIILGLN](SEQ ID NO:19)，是针对特定的小鼠CD4+T细胞。这些肽库或单肽不同于GAG mosaic 1,2或二价HIV嵌合Gag抗原(mosaic)，它们之间不是100％一致，从而能反映出本发明的疫苗的确诱导出了广谱针对三个病毒亚型的T细胞免疫反应。
结果如图6所示，显示出二价HIV嵌合Gag抗原(mosaic)的疫苗(pVAX-huPD1-mosaic)在小鼠模型中诱导出高水平、广谱的针对中国三种亚型(AE，B，C)的T细胞反应，同时证明了可溶性的PD1具有增强二价HIV嵌合Gag抗原的免疫原性。图中三个病毒亚型肽库的来源如上所述。
实施例5：恒河猴(Rhesus macaque)实验
本实施例中使用的活体动物实验经过了佛山科学技术学院兽医实验动物伦理委员会批准。免疫实验采用四只恒河猴，分别在第0，6，12，25周进行免疫(图7A)，每次免疫采用10-20mg/kg克他命进行麻醉，接着用4针电极阵列进行总共2mg二价pVAX-huPD1-mosaic核酸疫苗(即实施例3制备得到的pVAX-huPD1-mosaic的表达质粒)的肌肉电转接种。
每隔两周，用克他命和甲苯噻嗪的混合物麻醉动物后，进行采血。从血液里分离PBMC和血浆，存储于-80℃，随后用MABTECH公司的试剂盒按照其说明书操作来检测疫苗免疫反应(ELIspot Assay for Monkey Interferon-γ，MABTECH,3421M-2A)。
结果分析：
(1)在恒河猴第一次免疫时(即免疫接种的第0天)，没有检测出特异性的针对Gag的CD8+T细胞反应。
(2)然而在第二次免疫后的两个星期(第八周)发现pVAX-huPD1-mosaic核酸疫苗诱导出了高水平的针对B亚型HIV-Gag P24的三个不重叠(no-overlapping)亚肽库PP1(肽库24-1)、PP2(肽库24-2)和PP3(肽库24-3)(Cat#8117,Lot#10080196,NIH AIDS ReagentProgram)的特异性T细胞反应(图7B)，纵坐标显示分泌的斑点能增加至约2000个/106PBMC(外周血单核细胞)，横坐标代表每只猴子(1至4)，在免疫后的不同时间(0,8,12,14,16,25,28周)进行检测。结果证明诱导出B亚型内广谱的特异性T细胞反应。
(3)随后，在第14周和第28周，二价pVAX-huPD1-mosaic核酸疫苗免疫，又诱导出了高水平的特异性针对上述B亚型HIV-Gag的三个亚肽库PP1、PP2和PP3的T细胞反应，证明免疫记忆也已经建立。
(4)在第25周加强免疫后的第28周，又对AE亚型、B和BC亚型的多肽库，同时进行了检测，包括了HIV-Gag P17一个肽库和P24的B亚型的三个亚肽库PP1、PP2和PP3，结果检测到对每个亚型的T细胞记忆免疫反应(图7C)，其中纵坐标代表106PBMC(外周血单核细胞)分泌IFN-γT细胞的斑点数，横坐标代表每只猴子(1至4)，对AE，B或BC亚型多肽库的反应测定，结果证明本发明的二价pVAX-huPD1-mosaic疫苗抗原也可以在灵长动物体内诱导出三个亚型间广谱的特异性T细胞反应。
(5)接着又进一步利用HIV-Gag的AE亚型的肽库分解分析(图7D)，纵坐标显示106PBMC(外周血单核细胞)分泌IFN-γT细胞的斑点数，横坐标代表每只猴子(1至4)针对单肽(例如75、76等代表我们肽库中AE亚型单一肽的号码)的T细胞反应。从图可知每只猴子所针对的单肽T细胞反应是不一样的,提示本发明的二价huPD1-mosaic疫苗抗原符合人体使用个体差异的实际情况。AE亚型单一肽的氨基酸序列如下：
Gag38：PRTLNAWVKVVEEKG(SEQ ID NO:20)
Gag41：EKGFNPEVIPMFSAL(SEQ ID NO:21)
Gag42：NPEVIPMFSALSEGA(SEQ ID NO:22)
Gag49:GHQAAMQMLKETINE(SEQ ID NO:23)
Gag50:AMQMLKETINEEAAD(SEQ ID NO:24)
Gag66:IYKRWIILGLNKIVR(SEQ ID NO:25)
Gag68:GLNKIVRMYSPVSIL(SEQ ID NO:26)
Gag75:VDRFYKTLRAEQATQ(SEQ ID NO:27)
Gag76:YKTLRAEQATQEVKN(SEQ ID NO:28)
实施例6：临床样品实验
本实施例研究二价HIV嵌合Gag抗原在HIV自然感染中的实际反应性能，合成了二价HIV嵌合Gag抗原的肽库Mosaic p41-1(SEQ ID NO:3)和Mosaic p41-2(SEQ ID NO:4)分别用于检测中国自然感染HIV病人的T细胞ELISPOT反应。检测采用MABTECH公司的货号为3420-2A的Human IFN-g ELISpotBASIC(ALP)检测试剂盒进行，步骤按照配套的试剂盒说明书进行。接受抗病毒联合药物治疗的病人PBMC(外周血单核细胞)样本数量n＝9，没有接受治疗的对照病人PBMC(外周血单核细胞)样本数量n＝17。
结果如图8所示，横坐标1-26分别代表每个病人，其中1-17代表没有接受治疗的对照病人，18-26代表接受抗病毒联合药物治疗的病人，纵坐标代表针对二价HIV嵌合Gag抗原的两个肽库Mosaic p41-1和Mosaic p41-2，106PBMC(外周血单核细胞)中分泌IFN-γ的T细胞的斑点数。
结果分析：
(1)在没有接受治疗对照病人中，有广泛的针对Mosaic p41-1肽库(13/17,76％)和Mosaic p41-2肽库(7/17,41％)的ELISPOT反应(以SFU>200计算)(SFU:spot-formingunit,斑点形成单位)，而总体反应率(对Mosaic p41-1或Mosaic p41-2有阳性反应)达到94.1％(16/17)，因此基于本发明的二价HIV嵌合Gag抗原的疫苗可以成为HIV预防和临床治疗的候选疫苗。
(2)在接受抗病毒联合药物治疗的病人中，体内没有可以检测到的病毒，T细胞ELISPOT反应率要低于没有接受治疗的对照病人，总体反应率只有22％(2/9)。由此可见，本发明的二价HIV嵌合Gag抗原疫苗可用来加强治疗后的病人的抗HIV的广谱T细胞免疫反应，以促成免疫治疗。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。
SEQUENCE LISTING
<110> 艾克尔免疫愈疗有限公司
<120> HIV嵌合抗原、制备方法及应用
<130> 16PA0176CN.B1
<160> 28
<170> PatentIn version 3.3
<210> 1
<211> 1089
<212> DNA
<213> human
<400> 1
atgggggcaa gagcctccgt gctgtctggc gggaaactgg acgcctggga gaagatccgg 60
ctgagaccag gaggcaagaa aaagtaccgc ctgaagcaca tcgtgtgggc atcccgcgaa 120
ctggagcgat tcgccctgaa cccaggactg ctggaaaccg cagagggatg ccagcagatc 180
attgagcagc tgcagtctac actgaaaact ggctccgagg aactgaagtc tctgtttaac 240
accatcgctg tgctgtggtg cgtgcatcag cgcattgacg tgaaggatac aaaagaggcc 300
ctggacaaga tcgaggaagt gcagaacaag tcacagcaga agactcagca ggccgctgca 360
ggaaccggaa gctcctctaa ggtgagccag aactatccca ttgtccagaa tgcacaggga 420
cagatggtgc accagccact gagccctcgg accctgaacg catgggtgaa agtggtcgag 480
gaaaagggct tcaatcctga agtcatccca atgtttagtg cactgtcaga gggggccaca 540
cctcaggatc tgaacatgat gctgaatatc gtggggggac atcaggccgc tatgcagatg 600
ctgaaggaaa ctattaatga ggaagcagca gagtgggacc gagtgcaccc agtccatgca 660
ggaccaatcc cacctggaca gattcgagaa ccacgaggat ccgatatcgc cggcaccaca 720
tctactctgc aggagcagat tgggtggatg accaacaatc cacccatccc tgtgggagac 780
atctacaaac gctggatcat tctgggcctg aacaagatcg tgcgaatgta tagcccagtc 840
tccatcctgg atattcggca gggaccaaaa gagcccttca gggactacgt ggatcgcttt 900
tataagacac tgagagcaga acaggccact caggaggtga aaaattggat gacagagact 960
ctgctggtcc agaacgccaa tcctgactgc aaatctattc tgaaggctct ggggaccgga 1020
gcaacactgg aggaaatgat gaccgcttgt cagggagtgg gaggaccagg acacaaggca 1080
agggtcctg 1089
<210> 2
<211> 1089
<212> DNA
<213> human
<400> 2
atgggcgccc gagccagcat cctgcgggga ggcaagctgg ataaatggga gaagattagg 60
ctgcgccccg ggggaaaaaa gcactacatg ctgaagcatc tggtgtgggc ttctcgggaa 120
ctggagagat tcgcagtcaa cccaggcctg ctggaaacca gtgaggggtg caaacagatc 180
attaagcagc tgcagcccgc tctgcagacc ggaacagagg aactgcgcag tctgtttaac 240
actgtggcca ccctgtactg cgtgcaccag cgaatcgaga tcaaggacac aaaggaggcc 300
ctggataaaa tcgaggaaga gcagaataag tccaaaaaga aagctcagca gacagctgca 360
gatactggaa acaattctca ggtgagtcag aactatccaa tcgtccagaa tctgcagggc 420
cagatggtgc accagcctat tagcccaaga accctgaacg cctgggtgaa agtggtcgaa 480
gagaaggctt tcagccccga agtcatccct atgtttaccg ccctgtccga gggagctaca 540
cctcaggacc tgaacaccat gctgaataca gtgggcgggc accaggctgc tatgcagatc 600
ctgaaggaca ctattaatga agaggcagcc gagtgggata ggctgcaccc agtgcatgca 660
ggaccagtcg ctcctggaca gatgagagaa cctaggggaa gtgatatcgc cggcactacc 720
tcaaacctgc aggagcagat tggctggatg acaagcaatc ctccaatccc cgtgggggaa 780
atctacaaaa gatggatcat tctgggactg aacaagatcg tgaggatgta ttcacctact 840
agcatcctgg acatcaagca ggggccaaag gagcccttca gagactatgt ggataggttc 900
tttaagaccc tgagagctga acaggcatcc caggacgtga aaaattggat gactgatacc 960
ctgctggtcc agaacgcaaa tcctgattgc aaaacaatcc tgaaggccct gggcccagct 1020
gcaactctgg aggagatgat gaccgcttgc cagggcgtgg gaggaccttc acataaagcc 1080
agagtgctg 1089
<210> 3
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<212> PRT
<213> human
<400> 3
Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp
1 5 10 15
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys
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His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro
35 40 45
Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile Glu Gln Leu
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Gln Ser Thr Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn
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Thr Ile Ala Val Leu Trp Cys Val His Gln Arg Ile Asp Val Lys Asp
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Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln
100 105 110
Gln Lys Thr Gln Gln Ala Ala Ala Gly Thr Gly Ser Ser Ser Lys Val
115 120 125
Ser Gln Asn Tyr Pro Ile Val Gln Asn Ala Gln Gly Gln Met Val His
130 135 140
Gln Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu
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Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser
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Glu Gly Ala Thr Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly
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Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu
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Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Pro
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Pro Gly Gln Ile Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr
225 230 235 240
Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile
245 250 255
Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270
Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly
275 280 285
Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu
290 295 300
Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr
305 310 315 320
Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Lys Ala
325 330 335
Leu Gly Thr Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly
340 345 350
Val Gly Gly Pro Gly His Lys Ala Arg Val Leu
355 360
<210> 4
<211> 363
<212> PRT
<213> human
<400> 4
Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Lys Trp
1 5 10 15
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys
20 25 30
His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro
35 40 45
Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Lys Gln Leu
50 55 60
Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Arg Ser Leu Phe Asn
65 70 75 80
Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Ile Lys Asp
85 90 95
Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys
100 105 110
Lys Lys Ala Gln Gln Thr Ala Ala Asp Thr Gly Asn Asn Ser Gln Val
115 120 125
Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His
130 135 140
Gln Pro Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu
145 150 155 160
Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser
165 170 175
Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly
180 185 190
Gly His Gln Ala Ala Met Gln Ile Leu Lys Asp Thr Ile Asn Glu Glu
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Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Val Ala
210 215 220
Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr
225 230 235 240
Ser Asn Leu Gln Glu Gln Ile Gly Trp Met Thr Ser Asn Pro Pro Ile
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Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270
Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Lys Gln Gly
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Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu
290 295 300
Arg Ala Glu Gln Ala Ser Gln Asp Val Lys Asn Trp Met Thr Asp Thr
305 310 315 320
Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala
325 330 335
Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly
340 345 350
Val Gly Gly Pro Ser His Lys Ala Arg Val Leu
355 360
<210> 5
<211> 2928
<212> DNA
<213> 人造序列
<400> 5
atggacgcca tgctgcgcgg actgtgctgc gtgctgctac tgtgcggcgc cgtgttcgtg 60
agccccagcc aggagatcca cgcccgattc aggagaggag ccagaggagg atccatgcag 120
attcctcagg ctccatggcc tgtggtgtgg gcagtgctgc agctgggatg gagaccagga 180
tggttcctgg actcccctga cagaccatgg aatcccccta cattttctcc tgcactgctg 240
gtggtgactg agggcgataa cgccaccttc acatgcagct tttccaacac ttctgaaagt 300
ttcgtcctga attggtacag gatgtcaccc agcaaccaga ctgacaagct ggccgctttt 360
cccgaagacc gctcccagcc tgggcaagat tgccgattcc gggtgacaca gctgcctaat 420
ggaagggact ttcacatgag tgtggtccgc gctcggagaa acgattcagg aacctatctg 480
tgtggcgcaa tcagcctggc ccctaagaca cagatcaagg agagcctgag agccgaactg 540
agggtgactg agaggcgcgc tgaagtccca accgcacatc cttccccatc tccccgacca 600
gcaggacagg aattccgggg aggcggggga agtggaggag gaggatccgg aggaggagga 660
agcatggggg caagagcctc cgtgctgtct ggcgggaaac tggacgcctg ggagaagatc 720
cggctgagac caggaggcaa gaaaaagtac cgcctgaagc acatcgtgtg ggcatcccgc 780
gaactggagc gattcgccct gaacccagga ctgctggaaa ccgcagaggg atgccagcag 840
atcattgagc agctgcagtc tacactgaaa actggctccg aggaactgaa gtctctgttt 900
aacaccatcg ctgtgctgtg gtgcgtgcat cagcgcattg acgtgaagga tacaaaagag 960
gccctggaca agatcgagga agtgcagaac aagtcacagc agaagactca gcaggccgct 1020
gcaggaaccg gaagctcctc taaggtgagc cagaactatc ccattgtcca gaatgcacag 1080
ggacagatgg tgcaccagcc actgagccct cggaccctga acgcatgggt gaaagtggtc 1140
gaggaaaagg gcttcaatcc tgaagtcatc ccaatgttta gtgcactgtc agagggggcc 1200
acacctcagg atctgaacat gatgctgaat atcgtggggg gacatcaggc cgctatgcag 1260
atgctgaagg aaactattaa tgaggaagca gcagagtggg accgagtgca cccagtccat 1320
gcaggaccaa tcccacctgg acagattcga gaaccacgag gatccgatat cgccggcacc 1380
acatctactc tgcaggagca gattgggtgg atgaccaaca atccacccat ccctgtggga 1440
gacatctaca aacgctggat cattctgggc ctgaacaaga tcgtgcgaat gtatagccca 1500
gtctccatcc tggatattcg gcagggacca aaagagccct tcagggacta cgtggatcgc 1560
ttttataaga cactgagagc agaacaggcc actcaggagg tgaaaaattg gatgacagag 1620
actctgctgg tccagaacgc caatcctgac tgcaaatcta ttctgaaggc tctggggacc 1680
ggagcaacac tggaggaaat gatgaccgct tgtcagggag tgggaggacc aggacacaag 1740
gcaagggtcc tgggaggcgg gggaagtgga ggaggaggat ccggaggagg aggaagcatg 1800
ggcgcccgag ccagcatcct gcggggaggc aagctggata aatgggagaa gattaggctg 1860
cgccccgggg gaaaaaagca ctacatgctg aagcatctgg tgtgggcttc tcgggaactg 1920
gagagattcg cagtcaaccc aggcctgctg gaaaccagtg aggggtgcaa acagatcatt 1980
aagcagctgc agcccgctct gcagaccgga acagaggaac tgcgcagtct gtttaacact 2040
gtggccaccc tgtactgcgt gcaccagcga atcgagatca aggacacaaa ggaggccctg 2100
gataaaatcg aggaagagca gaataagtcc aaaaagaaag ctcagcagac agctgcagat 2160
actggaaaca attctcaggt gagtcagaac tatccaatcg tccagaatct gcagggccag 2220
atggtgcacc agcctattag cccaagaacc ctgaacgcct gggtgaaagt ggtcgaagag 2280
aaggctttca gccccgaagt catccctatg tttaccgccc tgtccgaggg agctacacct 2340
caggacctga acaccatgct gaatacagtg ggcgggcacc aggctgctat gcagatcctg 2400
aaggacacta ttaatgaaga ggcagccgag tgggataggc tgcacccagt gcatgcagga 2460
ccagtcgctc ctggacagat gagagaacct aggggaagtg atatcgccgg cactacctca 2520
aacctgcagg agcagattgg ctggatgaca agcaatcctc caatccccgt gggggaaatc 2580
tacaaaagat ggatcattct gggactgaac aagatcgtga ggatgtattc acctactagc 2640
atcctggaca tcaagcaggg gccaaaggag cccttcagag actatgtgga taggttcttt 2700
aagaccctga gagctgaaca ggcatcccag gacgtgaaaa attggatgac tgataccctg 2760
ctggtccaga acgcaaatcc tgattgcaaa acaatcctga aggccctggg cccagctgca 2820
actctggagg agatgatgac cgcttgccag ggcgtgggag gaccttcaca taaagccaga 2880
gtgctgtgat aaccgctcga gcggccggcg cgccgtttaa acaaagct 2928
<210> 6
<211> 495
<212> DNA
<213> human
<400> 6
atgcagattc ctcaggctcc atggcctgtg gtgtgggcag tgctgcagct gggatggaga 60
ccaggatggt tcctggactc ccctgacaga ccatggaatc cccctacatt ttctcctgca 120
ctgctggtgg tgactgaggg cgataacgcc accttcacat gcagcttttc caacacttct 180
gaaagtttcg tcctgaattg gtacaggatg tcacccagca accagactga caagctggcc 240
gcttttcccg aagaccgctc ccagcctggg caagattgcc gattccgggt gacacagctg 300
cctaatggaa gggactttca catgagtgtg gtccgcgctc ggagaaacga ttcaggaacc 360
tatctgtgtg gcgcaatcag cctggcccct aagacacaga tcaaggagag cctgagagcc 420
gaactgaggg tgactgagag gcgcgctgaa gtcccaaccg cacatccttc cccatctccc 480
cgaccagcag gacag 495
<210> 7
<211> 45
<212> DNA
<213> 人造序列
<400> 7
ggaggcgggg gaagtggagg aggaggatcc ggaggaggag gaagc 45
<210> 8
<211> 75
<212> DNA
<213> 人造序列
<400> 8
ccggaattcc ggggaggcgg gggaagtgga ggaggaggat ccggaggagg aggaagcatg
ggggcaagag cctcc 75
<210> 9
<211> 60
<212> DNA
<213> 人造序列
<400> 9
ccggcgcgcc gtttaaacaa agctccgctc gagcggttat cacagcactc tggctttatg 60
<210> 10
<211> 9
<212> PRT
<213> 人造序列
<400> 10
Ala Met Gln Met Leu Lys Asp Thr Ile
1 5
<210> 11
<211> 20
<212> PRT
<213> 人造序列
<400> 11
Thr Ser Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile
1 5 10 15
Ile Leu Gly Leu
20
<210> 12
<211> 135
<212> PRT
<213> Human
<400> 12
Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp
1 5 10 15
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys
20 25 30
His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro
35 40 45
Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Gly Cys Gln Gln Leu
50 55 60
Gln Ser Thr Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn
65 70 75 80
Thr Val Ala Thr Leu Trp Cys Val His Gln Arg Ile Asp Val Lys Asp
85 90 95
Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln
100 105 110
Gln Lys Thr Gln Gln Ala Ala Ala Gly Thr Gly Ser Ser Ser Lys Val
115 120 125
Ser Gln Asn Tyr Pro Ile Val
130 135
<210> 13
<211> 229
<212> PRT
<213> human
<400> 13
Gln Asn Ala Gln Gly Gln Met Val His Gln Pro Val Ser Pro Arg Thr
1 5 10 15
Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Gly Phe Asn Pro Glu
20 25 30
Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp
35 40 45
Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala Ala Met Gln
50 55 60
Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Asp Trp Asp Arg Thr
65 70 75 80
Trp Asp Arg Thr Ala Gly Pro Ile Pro Pro Gly Gln Ile Arg Glu Pro
85 90 95
Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile
100 105 110
Ala Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys
115 120 125
Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro
130 135 140
Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg
145 150 155 160
Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr
165 170 175
Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala
180 185 190
Asn Pro Asp Cys Lys Ser Ile Leu Lys Ala Leu Gly Thr Gly Ala Thr
195 200 205
Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Ser His
210 215 220
Lys Ala Arg Val Leu
225
<210> 14
<211> 16
<212> PRT
<213> 人造序列
<400> 14
Gly His Ala Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu
1 5 10 15
<210> 15
<211> 21
<212> PRT
<213> 人造序列
<400> 15
Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile
1 5 10 15
Ile Leu Gly Leu Asn
20
<210> 16
<211> 15
<212> PRT
<213> 人造序列
<400> 16
Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn
1 5 10 15
<210> 17
<211> 20
<212> PRT
<213> 人造序列
<400> 17
Thr Ser Asn Pro Pro Val Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile
1 5 10 15
Ile Leu Gly Leu
20
<210> 18
<211> 15
<212> PRT
<213> 人造序列
<400> 18
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu
1 5 10 15
<210> 19
<211> 21
<212> PRT
<213> 人造序列
<400> 19
Thr Asn Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile
1 5 10 15
Ile Leu Gly Leu Asn
20
<210> 10
<211> 15
<212> PRT
<213> 人造序列
<400> 10
Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Gly
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213> 人造序列
<400> 21
Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu
1 5 10 15
<210> 22
<211> 15
<212> PRT
<213> 人造序列
<400> 22
Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala
1 5 10 15
<210> 23
<211> 15
<212> PRT
<213> 人造序列
<400> 23
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu
1 5 10 15
<210> 24
<211> 15
<212> PRT
<213> 人造序列
<400> 24
Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Asp
1 5 10 15
<210> 25
<211> 15
<212> PRT
<213> 人造序列
<400> 25
Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg
1 5 10 15
<210> 26
<211> 15
<212> PRT
<213> 人造序列
<400> 26
Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu
1 5 10 15
<210> 27
<211> 15
<212> PRT
<213> 人造序列
<400> 27
Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln
1 5 10 15
<210> 28
<211> 15
<212> PRT
<213> 人造序列
<400> 28
Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn
1 5 10 15
Claims (10)
1.一种核酸，其特征为，所述核酸的序列选自：
1)包含SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列；或
2)核酸的同源序列，其与SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列有至少70％-99％的同一性；或
3)核酸的变体，其与SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列相比具有至少一个核苷酸的取代、缺失和/或添加；或
4)核酸的衍生物，其为1)-3)所述的核酸的序列的变体。
2.根据权利要求1所述的核酸，其特征为，进一步包含可溶性PD1序列；
任选的，所述可溶性PD1序列为人类可溶性PD1序列；
任选的，所述的人类可溶性PD1序列为SEQ ID NO:6所示的核苷酸序列；
任选的，所述的核酸序列进一步包含作为链接体的linker序列；
任选的，所述linker序列位于SEQ ID NO:1和SEQ ID NO:2所示的核苷酸序列之间；
任选的，所述linker序列位于SEQ ID NO:6和SEQ ID NO:1所示的核苷酸序列之间；
任选的，所述linker序列为SEQ ID NO:7所示的核苷酸序列；
任选的，所述位于SEQ ID NO:6和SEQ ID NO:1所示的核苷酸序列之间的linker序列与SEQ ID NO:6之间含有第一酶切位点序列；
任选的，所述第一酶切位点序列为EcoRI酶切序列；
任选的，所述的核酸序列进一步包含第二酶切位点序列；
任选的，所述第二酶切位点序列与SEQ ID NO:6相连；
任选的，所述第二酶切位点序列为BamHI酶切序列；
任选的，所述的核酸序列进一步包含组织纤溶酶原激活蛋白表达及释放的信号肽序列和起始密码子；
任选的，所述BamHI酶切序列依次与组织纤溶酶原激活蛋白表达及释放的信号肽序列和起始密码子相连接；
任选的，所述的核酸序列进一步包含第三酶切位点序列和第四酶切位点序列；
任选的，所述SEQ ID NO:2所示的核苷酸序列依次与终止密码子、第三酶切位点序列和第四酶切位点序列相连接；
任选的，所述的第三酶切位点序列和第四酶切位点序列分别为XhoI酶切序列和PmeI酶切序列；
任选的，所述的核酸序列为SEQ ID NO:5序列。
3.一种氨基酸，其特征为，由权利要求1或2所述的核酸序列编码。
4.一种载体，其特征为，含有权利要求1或2所述的核酸序列。
5.一种宿主细胞，其特征为，转化或转染了权利要求4所述的载体。
6.一种疫苗，其特征为，包含权利要求1或2所述的核酸序列、或者权利要求3所述的氨基酸序列、或者权利要求4所述的载体、或者权利要求5所述的宿主细胞。
7.根据权利要求6所述的疫苗，其特征为，还包含其他HIV疫苗；
任选的，所述其他HIV疫苗为基于PD1的疫苗；
任选的，还包含佐剂。
8.一种疫苗的制备方法，其特征为，包含制备权利要求6或7所述疫苗所需的步骤。
9.权利要求1或2所述的核酸序列、或者权利要求3所述的氨基酸序列、或者权利要求4所述的载体、或者权利要求5所述的宿主细胞在制备预防和治疗HIV疾病的药物的应用。
10.权利要求1或2所述的核酸序列、或者权利要求3所述的氨基酸序列、或者权利要求4所述的载体、或者权利要求5所述的宿主细胞在制备HIV疾病诊断试剂中的应用。
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| GARBER,D. ET AL.: "Synthetic construct HIV-1 Clade B consensus gag protein gene, complete cds GenBank: AY531263.1", 《GENBANK》 * |
| JINGYING ZHOU ET AL.: "PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
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