CN108192876A - 一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 - Google Patents
一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 Download PDFInfo
- Publication number
- CN108192876A CN108192876A CN201711288573.9A CN201711288573A CN108192876A CN 108192876 A CN108192876 A CN 108192876A CN 201711288573 A CN201711288573 A CN 201711288573A CN 108192876 A CN108192876 A CN 108192876A
- Authority
- CN
- China
- Prior art keywords
- adenovirus
- neutralizing epitope
- human type
- epitope
- hexon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000701161 unidentified adenovirus Species 0.000 title abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 108090000565 Capsid Proteins Proteins 0.000 title abstract description 4
- 102100023321 Ceruloplasmin Human genes 0.000 title abstract description 4
- 229940023143 protein vaccine Drugs 0.000 title 1
- 230000003472 neutralizing effect Effects 0.000 abstract description 27
- 241000700605 Viruses Species 0.000 abstract description 20
- 229960005486 vaccine Drugs 0.000 abstract description 15
- 208000015181 infectious disease Diseases 0.000 abstract description 11
- 229940125575 vaccine candidate Drugs 0.000 abstract description 6
- 230000028993 immune response Effects 0.000 abstract description 5
- 108700026758 Adenovirus hexon capsid Proteins 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 241001429382 Coxsackievirus A16 Species 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 9
- 241001529459 Enterovirus A71 Species 0.000 description 8
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 8
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 8
- 101000666856 Homo sapiens Vasoactive intestinal polypeptide receptor 1 Proteins 0.000 description 8
- NTIZESTWPVYFNL-UHFFFAOYSA-N Methyl isobutyl ketone Chemical compound CC(C)CC(C)=O NTIZESTWPVYFNL-UHFFFAOYSA-N 0.000 description 8
- 102100038388 Vasoactive intestinal polypeptide receptor 1 Human genes 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000003501 vero cell Anatomy 0.000 description 4
- 101710197658 Capsid protein VP1 Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 208000009525 Myocarditis Diseases 0.000 description 3
- 210000000234 capsid Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229940023041 peptide vaccine Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229940031626 subunit vaccine Drugs 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 208000006740 Aseptic Meningitis Diseases 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 241000193096 Human adenovirus B3 Species 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 206010027201 Meningitis aseptic Diseases 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 101710106388 Structural protein VP1 Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Classifications
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
-
- 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
- C12N15/86—Viral vectors
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及重组病毒基因工程技术领域，具体涉及一种人3型腺病毒衣壳蛋白同时展示CA16双中和抗原表位疫苗候选株的制备方法和应用，在人3型腺病毒六邻体上的两个高变区同时插入CA16的中和表位。该疫苗候选可以提高CA16中和抗原表位的抗原性，诱导抗CA16感染的免疫反应，利用该疫苗候选株可以制备预防CA16感染的重组表位疫苗。
Description
技术领域
本发明涉及重组病毒基因工程技术领域，具体涉及一种人3型腺病毒衣壳蛋白同时展示CA16双中和抗原表位疫苗候选株的制备方法和应用。
背景技术
手足口病是由肠道病毒引起的急性传染病，该病主要感染5岁以下婴幼儿，可引发手、足、口腔等部位的疱疹，少数患儿可引起心肌炎、肺水肿、无菌性脑膜脑炎等并发症。柯萨奇A16型与肠道病毒71型是引起手足口病的常见病原。CA16与EV71呈交替流行或者同时在一个地区内流行。在以往的研究中，大多数手足口的致死病例是由EV71感染引起的，所以人们对EV71的研究比较多。但通过对近些年的手足口病原的检测及报道，CVA16感染也可能导致严重的手足口病，像无菌性脑膜炎，心肌炎，肺部综合征，严重甚至发生致死性心肌炎和肺炎。此外，EV71和CA16的混合感染会加重患者病情，使手足口病更加难以控制。
疫苗的使用可以有效的控制病毒传播。目前，预防EV71的疫苗已经上市，由于EV71疫苗的使用，在一定程度上使得的重症手足口病的发生率有所下降。但目前由CVA16感染引起的重症手足口病还没有商品化的疫苗投入使用，这使得人们对重症手足口病的预防还没有得到完全控制，婴幼儿仍然会因手足口病而发生相应的重症。如果预防CVA16的疫苗在市面上大量使用，那么势必会更加降低手足口病的重症情况。然而，相比于EV71，CVA16的基因功能尚未完全明确，无法确定其病毒毒性基因，因此目前市面上还没有有效的CVA16减毒疫苗株，并且减毒活疫苗可能毒力返强，存在一定安全问题。随着分子生物学的研究进展，人们对病毒结构的不断解析，利用重组技术研究亚单位疫苗可以有效的提高疫苗的安全性。CVA16为正链小RNA病毒，病毒颗粒的空间结构为二十面体，由内部核酸以及衣壳构成，无囊膜结构，其开放阅读框P1区编码结构蛋白，分为VP1，VP2，VP3，VP4四个部分。CVA16 VP1上集中了主要抗原决定簇。目前已经研制CVA16病毒样颗粒(VLP)，并且在小鼠中进行实验，结果表明CVA16 VLP亚单位疫苗可以诱导出较强的免疫反应[17]。但由于VLPs产量较低，生产成本高，且在规模生产时，易导致产品结构和组分不相容，限制了其在疫苗研究中的发展。
表位疫苗是近年来发展起来的一种新型疫苗，相对传统疫苗，拥有较多优势。病毒抗原片段上的中和抗原表位可以直接刺激机体产生中和抗体。目前已经研究众多的合成肽疫苗预防不同疾病[19-21]。CVA16的中和抗原表位主要为其结构蛋白VP1上，其中和抗原表位可以刺激机体产生中和抗体。虽然合成肽疫苗具有许多优点，但其仍存在许多问题。合成肽的免疫原性较低，抗原表位的筛选技术不成熟，免疫时需要使用免疫佐剂，病原微生物的中和表位多为构像表位，这些难题阻碍合成肽疫苗的发展。
腺病毒载体高表达，使用其作为疫苗载体无需添加佐剂，可通过肌肉或粘膜方式免疫，免疫后的机体可以同时诱导B细胞与T细胞免疫反应，易于生产制备。因此，腺病毒载体被广泛应用于各种预防性和治疗性疫苗的研发。腺病毒六邻体的高变区可以被外源氨基酸替换，作为嵌合疫苗载体，从而使机体直接针对所递呈的片段而产生抗体。随着人们对腺病毒蛋白功能的不断研究，目前已发现众多型别腺病毒可以作为嵌合疫苗载体。人3型腺病毒作为B组腺病毒的一种，其与CA16具有相同的受体。
发明内容
为了克服现有技术中存在的缺点和不足，本发明的目的在于提供一种人3型腺病毒衣壳蛋白同时展示CA16双中和抗原表位疫苗候选株的制备方法和应用。
本发明提供了一种重组人3型腺病毒，人3型腺病毒六邻体的HVR1、HVR2区上同时插入CA16的SP55的中和表位抗原表位，所述CA16中和表位SP55的氨基酸序列为PKPTSRDSFAWQTAT。
优选的，所述CA16中和表位SP55的替换区为HAdv3六邻体的HVR1和HVR2区。
更为优选的，所述CA16SP55中和表位替换氨基酸序列为Ad3六邻体的HVR1区的AGEER及HVR2区的TTEGEE。
本发明还提供了一种人3型腺病毒-CA16的双中和表位疫苗候选株，所述双中和表位苗候选株含有权利要求1-3任一项所述的重组人3型腺病毒。
本发明还提供了一种载体，所述载体包含上述所述的重组人3型腺病毒。
本发明还提供了一种重组人3型腺病毒的制备方法，包括如下步骤：
在人3型腺病毒的六邻体上的HVR1区及HVR2区同时嵌入CA16中和表位SP55；其中，
所述CA16中和表位SP55的氨基酸序列为PKPTSRDSFAWQTAT。
所述CA16中和表位SP55所替换氨基酸为Ad3六邻体的HVR1区的AGEER及HVR2区的TTEGEE。
本发明所提供的重组腺病毒，允许CA16中和表位个别氨基酸的缺失、替换或添加。但需要保持中和表位的免疫原性；允许被置换的HVR1及HVR2区个别氨基酸的缺失、替换或添加，但需要保持腺病毒的稳定性，同时保证外源表位的免疫活性；本发明中嵌入的CA16表位不限于SP55，也可以是其他的保护性T细胞表位或B细胞表位，但需要保持腺病毒的稳定性和保证外源表位的免疫原性。
本发明的有益效果在于：本本发明中，在六邻体衣壳上同时展示CA16中和表位，该重组腺病毒可以产生抗CA16感染的免疫应答，多次免疫注射能加强免疫，还可以预防CA16感染。
本发明通过采用人3型腺病毒的衣壳展示CVA16的中和抗原表位，将CA16的SP55的中和表位抗原表位同时插入到人3型腺病毒六邻体的HVR1、HVR2区，构建同时展示CVA16相同表位的嵌合病毒，为预防CVA16感染奠定基础，为更好的防治重症手足口病提供借鉴工具。
附图说明
图1是抗AdSP55R1R2△E3GFP血清对CA16感染VERO细胞图片；其中，(A)为AdSP55R1R2△E3GFP免疫血清32倍稀释后与100TCID50的CA16 37℃混合孵育后接种VERO后的细胞图片；(B)为100TCID50的CA16 37℃混合孵育后接种VERO72h后的细胞图片。
具体实施方式
为了便于本领域技术人员的理解，下面结合实施例及附图1对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，下列实施例中未提及的具体实验方法，通常按照常规实验方法进行。
实施例1：构建重组病毒载体
以HAdV3载体为模版，重叠PCR扩增含有CA16病毒主要中和抗原表位的六邻体片段，将CA16的中和抗原表位替换腺病毒六邻体上HVR1区的氨基酸序列。经过Cla I，Bam H I双酶切后与经过Cla I，Bam H I酶切后的穿梭质粒PBRLOR连接构建穿梭质粒，命名为PBRLOR-CA16HVR1。在按照相同的方法，将同样氨基酸序列的CA16中和抗原表位替换HVR2区的氨基酸，构建PBRLOR-CA16HVR1-HVR2。将构建好的穿梭质粒经过EcoR I，Sal I双酶切与骨架质粒pBRAd3△E3GFP经过Pac I，AvR II在BJ5183内同源重组，得到重组质粒pAdSP55HVR1-HVR2△E3GFP。所使用的引物见表1，CA16r1，CA16r2为PCR筛选鉴定用特异引物。
表1重叠PCR法扩增外源表位嵌入的HAdv3六邻体基因使用的引物
将上述重组得到的质粒pAdSP55HVR1-HVR2△E3GFP经过AsisI酶切线性化后转染AD293细胞，拯救包装得到重组病毒。大量培养并CsCl密度梯度离心纯化重组病毒粒子。经过纯化的病毒粒子达到1×1012VPs/ml，测定OD260/OD280比值约1.2-1.4，内毒素检测<10EU/ml。重组病毒AdSP55R1R2△E3GFP在HEp-2细胞中连续传代10代后提取病毒基因组进行酶切鉴定、测序鉴定，表明重组病毒基因组保持稳定，没有基因缺少、重组现象。
实施例2：重组病毒的免疫原性实验
制备抗血清，进行血清中和试验，验证重组病毒载体的免疫原性。将纯化的重组病毒AdSP55R1R2△E3GFP肌肉注射免疫小鼠，得到多克隆血清，然后进行微量中和试验，以检测重组病毒产生的抗体是否具有体外中和CA16病毒的能力。
免疫3次后第14天采集的小鼠血清中和反应的实验结果见表2，表2为多次免疫小鼠血清中和效价，实验结果数据表明多次免疫后抗CA16病毒的免疫应答得到加强。
表2单次免疫血清中和效价
抗AdSP55R1R2△E3GFP血清对CA16感染VERO细胞的阻断实验结果如图1所示，从图1可以看出，使用AdSP55R1R2△E3GFP病毒多次免疫小鼠后，产生的血清可以在VERO细胞水平上经过32倍稀释后，可以完全阻断100TCID50的CA16感染。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (6)
1.一种重组人3型腺病毒，其特征在于：人3型腺病毒六邻体的HVR1、HVR2区上同时插入CA16的SP55的中和表位抗原表位，所述CA16中和表位SP55的氨基酸序列为PKPTSRDSFAWQTAT。
2.根据权利要求1所述的一种重组人3型腺病毒，其特征在于：所述CA16中和表位SP55的替换区为HAdv3六邻体的HVR1和HVR2区。
3.根据权利要求1所述的一种重组人3型腺病毒，其特征在于：所述CA16SP55中和表位替换氨基酸序列为Ad3六邻体的HVR1区的AGEER及HVR2区的TTEGEE。
4.一种人3型腺病毒-CA16的双中和表位疫苗候选株，其特征在于：所述双中和表位苗候选株含有权利要求1-3任一项所述的重组人3型腺病毒。
5.一种载体，其特征在于：所述载体包含权利要求1-3任一项所述的重组人3型腺病毒。
6.如权利要求1-3任一项所述的一种重组人3型腺病毒的制备方法，其特征在于：包括如下步骤：
在人3型腺病毒的六邻体上的HVR1区及HVR2区同时嵌入CA16中和表位SP55；其中，
所述CA16中和表位SP55的氨基酸序列为PKPTSRDSFAWQTAT。
所述CA16中和表位SP55所替换氨基酸为Ad3六邻体的HVR1区的AGEER及HVR2区的TTEGEE。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711288573.9A CN108192876A (zh) | 2017-12-07 | 2017-12-07 | 一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711288573.9A CN108192876A (zh) | 2017-12-07 | 2017-12-07 | 一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108192876A true CN108192876A (zh) | 2018-06-22 |
Family
ID=62573640
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711288573.9A Pending CN108192876A (zh) | 2017-12-07 | 2017-12-07 | 一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108192876A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283795A (zh) * | 2019-05-30 | 2019-09-27 | 广州市妇女儿童医疗中心 | 同时展示ev71病毒和cva16病毒中和抗原表位的重组腺病毒 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011022522A1 (en) * | 2009-08-18 | 2011-02-24 | Takayuki Shiratsuchi | Modification of recombinant adenovirus with immunogenic plasmodium circumsporozoite protein epitopes |
| CN105219740A (zh) * | 2015-09-29 | 2016-01-06 | 东莞市第八人民医院 | 一种重组人3型腺病毒及制备方法和应用 |
-
2017
- 2017-12-07 CN CN201711288573.9A patent/CN108192876A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011022522A1 (en) * | 2009-08-18 | 2011-02-24 | Takayuki Shiratsuchi | Modification of recombinant adenovirus with immunogenic plasmodium circumsporozoite protein epitopes |
| CN105219740A (zh) * | 2015-09-29 | 2016-01-06 | 东莞市第八人民医院 | 一种重组人3型腺病毒及制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| 孙一晟等: "PCV-2 ORF2和PRRSV GP5双基因共表达重组腺病毒的构建及小鼠免疫试验", 《动物医学进展。》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283795A (zh) * | 2019-05-30 | 2019-09-27 | 广州市妇女儿童医疗中心 | 同时展示ev71病毒和cva16病毒中和抗原表位的重组腺病毒 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10004796B2 (en) | Adenoviral vector-based vaccine against enterovirus infection | |
| CN106661102B (zh) | 作为对抗肠病毒感染之免疫原的病毒颗粒及其制造 | |
| Zhang et al. | Vaccination with coxsackievirus B3 virus-like particles elicits humoral immune response and protects mice against myocarditis | |
| Zhang et al. | Enterovirus D68 virus-like particles expressed in Pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice | |
| Liu et al. | Immunological and biochemical characterizations of coxsackievirus A6 and A10 viral particles | |
| CN103966263A (zh) | 一种重组人3型腺病毒及其制备方法和应用 | |
| Tsou et al. | Recombinant adeno-vaccine expressing enterovirus 71-like particles against hand, foot, and mouth disease | |
| Mattion et al. | Attenuated poliovirus strain as a live vector: expression of regions of rotavirus outer capsid protein VP7 by using recombinant Sabin 3 viruses | |
| CN102614507B (zh) | 一种o型口蹄疫分子标记疫苗及其制备方法 | |
| CN117187265B (zh) | 重组柯萨奇病毒a10 vlp及其用途 | |
| CN105219740A (zh) | 一种重组人3型腺病毒及制备方法和应用 | |
| CN106244602A (zh) | 一种携带两个g基因和vp2基因的重组狂犬病病毒及其应用 | |
| Zhang et al. | Coxsackievirus B5 virus-like particle vaccine exhibits greater immunogenicity and immunoprotection than its inactivated counterpart in mice | |
| JP2021500880A (ja) | ２つの発現カセットを有するサルアデノウイルスベクター | |
| CN103966174B (zh) | 一种重组人3型腺病毒及其制备方法和应用 | |
| Zhang et al. | Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71 | |
| CN105238766B (zh) | 人3型腺病毒展示人14型腺病毒中和抗原表位疫苗候选株的制备方法和应用 | |
| CN105238767B (zh) | 人3型腺病毒展示人55型腺病毒中和抗原表位疫苗候选株的制备方法和应用 | |
| CN108514635A (zh) | 重组三价腺病毒疫苗及其构建方法 | |
| CN103865923B (zh) | 流感病毒为载体的HAdV嵌合疫苗的制备及其应用 | |
| Chi et al. | A nucleoside-modified mRNA vaccine prevents enterovirus A71 infection in mouse model | |
| CN108192876A (zh) | 一种人3型腺病毒衣壳蛋白同时展示ca16双中和抗原表位疫苗候选株的制备方法和应用 | |
| CN106906186B (zh) | 一种同时预防人3型腺病毒-人55型腺病毒-人7型腺病毒的多表位疫苗候选株 | |
| CN103757032A (zh) | 一种以流感病毒为载体的hcv嵌合疫苗及其制备方法 | |
| Lyu et al. | Achievements, challenges and prospects for the development of broadly protective multivalent vaccines and therapeutic antibodies against hand, foot and mouth disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |