CN113820495B - 新冠病毒包膜蛋白中和抗体活性评价方法 - Google Patents
新冠病毒包膜蛋白中和抗体活性评价方法 Download PDFInfo
- Publication number
- CN113820495B CN113820495B CN202110989583.5A CN202110989583A CN113820495B CN 113820495 B CN113820495 B CN 113820495B CN 202110989583 A CN202110989583 A CN 202110989583A CN 113820495 B CN113820495 B CN 113820495B
- Authority
- CN
- China
- Prior art keywords
- cells
- pseudovirus
- novel coronavirus
- protein
- hiv
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000003472 neutralizing effect Effects 0.000 title claims abstract description 21
- 230000000694 effects Effects 0.000 title claims abstract description 18
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 14
- 101710091045 Envelope protein Proteins 0.000 title claims description 5
- 101710188315 Protein X Proteins 0.000 title claims description 5
- 102100021696 Syncytin-1 Human genes 0.000 title 1
- 241001112090 Pseudovirus Species 0.000 claims abstract description 45
- 229960005486 vaccine Drugs 0.000 claims abstract description 9
- 108010022999 Serine Proteases Proteins 0.000 claims abstract description 5
- 102000012479 Serine Proteases Human genes 0.000 claims abstract description 5
- 102100031673 Corneodesmosin Human genes 0.000 claims description 19
- 101710139375 Corneodesmosin Proteins 0.000 claims description 19
- 239000013612 plasmid Substances 0.000 claims description 13
- 239000013613 expression plasmid Substances 0.000 claims description 10
- 108700002856 Coronavirus Envelope Proteins Proteins 0.000 claims description 9
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 claims description 4
- 238000002405 diagnostic procedure Methods 0.000 claims description 2
- 239000013598 vector Substances 0.000 abstract description 4
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 abstract 1
- 238000007435 diagnostic evaluation Methods 0.000 abstract 1
- 102000048657 human ACE2 Human genes 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 48
- 241000700605 Viruses Species 0.000 description 26
- 241001678559 COVID-19 virus Species 0.000 description 14
- 108020003175 receptors Proteins 0.000 description 14
- 102000005962 receptors Human genes 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 11
- 230000009545 invasion Effects 0.000 description 11
- 239000004098 Tetracycline Substances 0.000 description 10
- 229960002180 tetracycline Drugs 0.000 description 10
- 229930101283 tetracycline Natural products 0.000 description 10
- 235000019364 tetracycline Nutrition 0.000 description 10
- 150000003522 tetracyclines Chemical class 0.000 description 10
- 101000638154 Homo sapiens Transmembrane protease serine 2 Proteins 0.000 description 8
- 238000006386 neutralization reaction Methods 0.000 description 8
- 108700008625 Reporter Genes Proteins 0.000 description 7
- 102100031989 Transmembrane protease serine 2 Human genes 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 5
- 208000025721 COVID-19 Diseases 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 3
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 3
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 3
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229940096437 Protein S Drugs 0.000 description 2
- 101710198474 Spike protein Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003405 preventing effect Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 230000005100 tissue tropism Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 229940022962 COVID-19 vaccine Drugs 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101710180643 Leishmanolysin Proteins 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 229940022005 RNA vaccine Drugs 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 208000037847 SARS-CoV-2-infection Diseases 0.000 description 1
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000026502 entry into host cell Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- -1 n Species 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002962 plaque-reduction assay Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 229940126583 recombinant protein vaccine Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229940126580 vector vaccine Drugs 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- 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
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
- C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本申请提供了一种新冠病毒中和抗体活性非诊断评价方法，其中使用了基于HIV/Lu c报告载体的假病毒以及跨膜丝氨酸蛋白酶瞬时转染的稳定表达人ACE2(hACE2)受体的细胞。本发明还提供了相应假病毒产品在评价新冠病毒疫苗效果中的应用。
Description
技术领域
本申请属于蛋白、疫苗和微生物领域。具体地，本申请提供了一种新冠病毒包膜蛋白中和抗体活性评价方法。
背景技术
SARS-CoV-2的S蛋白介导病毒吸附和侵入细胞的过程，是诱导中和抗体的主要抗原。目前被广泛应用于评估患者体内或者疫苗接种人群接种后产生的保护性免疫应答程度的方法包括ELISA等技术在内的免疫学检测方法,检测的靶标主要是针对S蛋白特定区域的结合抗体而非真正具有阻止病毒侵染活性的中和抗体，因此难以真实评价抗体对病毒的中和作用以及对宿主的保护效果。评估SARS-CoV-2疫苗诱导的中和抗体的最直接的方法是使用活病毒的感染中和试验或空斑减数试验，但是活病毒的操作必须在BSL-3实验室中进行，受实验条件和病毒来源等因素的限制。另外,由于活病毒在扩增和传代过程中易发生基因突变且培养条件和结果判读标准的不同，不同实验室的活病毒检测结果常常存在较大差异。在目前病毒变异频繁，各种疫苗集中启用的情况下，急需寻找一种准确检测中和抗体活性的方法。
发明内容
为了解决SARS-CoV-2活病毒应用于相关分析试验中存在种种局限问题，本研究建立了一种基于HIV/Luc报告载体的假病毒检测系统用于SARS-CoV-2中和抗体的检测和定量分析，操作相对安全，检测结果更加稳定。将经过密码子优化的且C末端缺失19个氨基酸的S蛋白插入假病毒颗粒中，假病毒将以与活病毒相同的方式进入细胞。本研究中构建的假病毒可用于评价所有类型的中和抗体以及针对S蛋白设计的小分子药物在阻止病毒进入细胞方面的效果。本研究通过将HIV骨架质粒与S蛋白表达质粒共转染293T细胞制备假病毒，同时用跨膜丝氨酸蛋白酶(TMPRSS2)瞬时转染的hACE2受体稳定表达T-REx 293细胞作为靶细胞评价假病毒进入的水平。TMPRSS2是一种可切割ACE2和刺突蛋白的细胞表面蛋白酶，在病毒入侵过程中可对冠状病毒的刺突蛋白预激活，为SARS-CoV-2进入细胞提供便利。本研究中构建的假病毒带有荧光素酶报告基因，可以通过荧光测定仪精确地检测报告基因的表达,实现病毒的定量检测。通过替换S蛋白表达质粒，可以研究抗S蛋白抗体针对不同突变株的交叉中和作用。该假病毒检测系统除了用于评估中和抗体效价和病毒进入抑制剂在阻断病毒入侵细胞过程中的效果外，它还可以用于研究病毒的组织嗜性和受体识别方式。
新冠病毒包膜蛋白中和抗体活性评价技术的实现包括：1.hACE2受体诱导表达靶细胞T-REx 293-hACE2的构建；2.通过在靶细胞T-REx 293-hACE2中同时表达TMPRSS2，对SARS-CoV-2假病毒预激活从而提高其感染量；3.基于HIV骨架的表达新冠病毒包膜蛋白假病毒单轮感染系统建立。
一方面，本申请提供了一种新冠病毒中和抗体活性非诊断评价方法，其中使用了基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒。
进一步地，所述基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒是通过将HIV/Luc骨架质粒与S蛋白或S蛋白突变体表达质粒共转染来制备的。
进一步地，共转染的对象为293T细胞。
进一步地，所述HIV/Luc骨架质粒为pNL4-3.Luc.R-E-。
进一步地，所述方法中还使用了稳定表达hACE2受体的T-REx 293细胞作为靶细胞。
进一步地，所述靶细胞以跨膜丝氨酸蛋白酶瞬时转染。
另一方面，本申请提供了一种基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒在评价新冠病毒疫苗效果中的应用。
进一步地，所述基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒是通过将HIV/Luc骨架质粒与不同S蛋白突变体表达质粒共转染293T细胞来制备的。
进一步地，所述HIV/Luc骨架质粒为pNL4-3.Luc.R-E-。
另一方面，本申请提供了上述假病毒在制备新冠病毒疫苗效果试剂组中的应用。
本申请的方法和假病毒可用于诊断或非诊断用途，优选地，用于新冠病毒相关科学研究特别是疫苗研究，疾控数据统计和研判等非诊断用途。
本申请中的抗体可以为各种疫苗，包括但不限于灭活疫苗、重组蛋白/亚单位疫苗、腺病毒载体疫苗、RNA疫苗等；pNL4-3.Luc.R-E-有市售产品，也可以根据相关文献构建。
本申请的基于假病毒侵染平台的新冠病毒中和抗体检测系统，具有安全性高，检测结果稳定性好，可重复性佳的优势,可通过将病毒与抗体共同孵育后加至表达hACE2+TMPRSS2的T-REx 293细胞中，培养48h后，通过检测荧光素酶报告基因的表达来评价抗体或者小分子抑制剂对病毒侵入宿主细胞的保护作用，用于评估中和抗体效价和病毒进入抑制剂在阻断病毒入侵细胞过程中的效果及病毒的组织嗜性和受体识别方式等机制研究。
附图说明
图1为HIV/Luc报告基因结构及基于HIV/Luc报告载体的假病毒包装示意图；
图2为感染模型的建立与SARS-CoV-2病毒进入宿主细胞的检测：左图为表达hACE2受体+TMPRSS2的T-REx 293靶细胞感染平台和病毒侵入系统构建示意图；右上为假病毒侵入实验(该假病毒系统带有EGFP报告基因，可用荧光显微镜检测)，Tet+表示加入四环素，Tet-表示未加四环素，表达hACE2受体和TMPRSS2的T-REx 293细胞可以有效支持SARS-CoV和SARS-CoV-2假病毒的侵入，却不能支持MERS-CoV假病毒侵入；右下为这三种假病毒侵入效率的直方图。
图3为CB6单克隆抗体中和检测结果。
具体实施方式
为让本领域的技术人员更加清晰直观地了解本发明，下面将结合附图，对本发明作进一步的说明。
实施例1hACE2受体诱导表达系统的建立和鉴定
(1)SARS-CoV-2假病毒感染靶细胞T-REx 293-hACE2+TMPRSS2
将hACE2受体序列克隆至诱导表达载体pCDNA5，同时在其氨基端引入myc标签，便于后续的表达鉴定。建立表达ACE2的人胚肾293细胞系(FLP-IN T-REx 293-hACE2)，利用四环素诱导ACE2的表达。通过控制四环素浓度，可间接调控ACE2的表达水平。ACE2受体诱导表达的同时瞬时表达TMPRSS2蛋白酶，从而对SARS-CoV-2病毒预激活，可以显著提高SARS-CoV-2感染能力。
(2)hACE2受体诱导表达系统的鉴定
T REx 293-hACE2细胞用四环素处理24h后，细胞裂解提取总蛋白，通过WesternBlot法鉴定受体分子的表达。
实施例2SARS-CoV-2S蛋白表达质粒的构建及假病毒的包装与体外感染模型的建立
(1)S蛋白表达质粒的构建
S蛋白作为包膜蛋白，在293T细胞中的表达水平较低，因此我们对SARS-CoV-2(MN908947)C末端缺失19个氨基酸的S蛋白在不改变氨基酸序列的前提下，进行密码子优化，克隆至pSecTag2/HygroA载体，扩增、纯化。
(2)包含S蛋白的假病毒的包装(HIV/Luc报告基因结构及基于HIV/Luc报告载体的假病毒包装如图1所示)
包含S蛋白的真核表达质粒通过与Env缺失的、带有荧光素酶报告基因的骨架质粒pNL4-3.Luc.R-E-共转染293T细胞，获得重组假病毒，具体方法如下：
以5×106个293T细胞接种10cm细胞培养皿，过夜培养后汇合度达到80％。
将包含S蛋白的表达质粒2.5μg和骨架质粒pNL4-3.Luc.R-E-7.5μg按照1：3(w/w)的比例加入含有500μL无抗生素无血清DMEM的1.5mL离心管中混匀。
加入30μL FuGENE HD(DNA与转染试剂1:3，w/v)，混匀后室温静置15-30min。将质粒-转染试剂混合液全部逐滴加入10cm细胞培养皿中，轻摇混匀。置于37℃，CO2培养箱培养。
培养48-72h后收获包含假病毒的培养上清，补加FBS至终浓度达到20％。使用0.45μm滤器过滤病毒液，分装后冻存于-80℃冰箱保存备用。
(3)建立SARS-CoV-2假病毒体外感染模型以及假病毒侵入实验(过程和结果见图2)
a)T-REx 293-hACE2诱导表达细胞在转染TMPRSS2前12h接种至6孔板，使得细胞在转染时丰度约80％。
b)转染pCAGGS-TMPRSS2质粒4μg至6孔板T-REx 293-hACE2细胞，转染24h后，接种至96孔细胞培养板，次日，利用四环素诱导表达hACE2受体，四环素(终浓度2μg/mL)诱导24h后，感染SARS-CoV-2假病毒颗粒，感染后48-72h检测细胞的荧光素酶活性。
(4)假病毒滴度测定
将转染了pCAGGS-TMPRSS2的T-REx 293-hACE2细胞接种至96孔板，加入2μg/mL四环素诱导24h后，对待测SARS-CoV-2进行滴定。首先对病毒原液进行10倍稀释，然后进行3倍比系列稀释，共9个梯度，每个梯度设置6个复孔。同时设置无假病毒的细胞对照。于37℃，5％CO2培养箱孵育48-72h，裂解细胞并加入荧光素酶底物进行化学发光检测。根据Reed-Muench法计算假病毒的50％组织培养感染剂量(TCID50)。
实施例3利用本申请的方法评价单克隆抗体中和能力
以单克隆抗体CB6(一种靶向与SARS-CoV-2S蛋白RBD区的单克隆抗体,Shi Rui,Shan Chao,Duan Xiaomin et al.Nature,2020,)为例，利用本申请的方法评价CB6单抗在抑制SARS-CoV-2S蛋白参考株及D614G突变株感染侵入宿主细胞的能力。
方法：
(1)包含SARS-CoV-2假病毒及其突变株(突变D614G)制备如前面所述。将2.5μgpCAGGS-SARS-CoV-2S和7.5μg pNL4-3.Luc.R-E-质粒共转染293T细胞，收获转染后48h和72h病毒上清，离心去除细胞碎片，通过0.45μm无菌膜过滤、分装，并测定TCID50。
(2)CB6单克隆抗体中和试验：T-REx 293-hACE2细胞铺于6孔板中，转染pCAGGS-TMPRSS2质粒，转染后24h按照2×104个/孔接种至96孔板，待其贴壁后每孔加入终浓度2μg/mL的四环素，诱导表达24h。含1000TCID50假病毒的100μL上清液与等体积的五倍系列稀释(浓度范围从0.64ng/mL到10μg/mL)的CB6抗体在37℃孵育1h。孵育结束后将假病毒与CB6单抗的混合液转移至预铺靶细胞的96孔细胞板中。于37℃ CO2培养箱再培养48-72h。弃去上清，每孔加入30μL细胞裂解液，作用10min后添加荧光素酶底物50μL/孔。荧光素酶活性测定使用GloMax96微孔板发光仪(Promega)。中和百分数的计算公式如下： 使用GraphPad Prism 6.0软件计算半数抑制浓度(IC50，即中和百分数为50％时对应的CB6抗体的浓度)，结果如图3所示。
实施例4利用本申请的方法对COVID-19患者血浆样本中和抗体水平检测
病毒制备及滴度测定如例1所述，对COVID-19患者血浆样本中和抗体水平检测方法如下：T-REx 293-hACE2细胞铺于6孔板中，转染pCAGGS-TMPRSS2质粒，转染后24h按照2×104个/孔接种至96孔板，待其贴壁后每孔加入终浓度2μg/mL的四环素，诱导表达24h。患者血浆样本于56℃作用1h灭活。含1000TCID50假病毒的100μL上清液与等体积的五倍系列稀释(初始稀释度为100)的灭活的患者血浆抗体在37℃孵育1h。孵育结束后将假病毒与患者血浆的混合液转移至预铺靶细胞的96孔细胞板中。于37℃ CO2培养箱再培养48-72h。弃去上清，每孔加入30μL细胞裂解液，作用10min后添加荧光素酶底物50μL/孔。荧光素酶活性测定使用GloMax96微孔板发光仪(Promega)。中和百分数的计算公式如下： 使用GraphPad Prism 6.0软件计算半数抑制剂量(ID50，即中和百分数为50％时对应的患者血浆样本稀释度)，结果如表1所示。
表1 COVID-19患者血浆中和抗体检测
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于这里的实施例，本领域技术人员根据本发明的揭示，对于本发明做出的改进和修改都应该在本发明的保护范围之内。
Claims (2)
1.一种新冠病毒中和抗体活性非诊断评价方法，其特征在于，所述方法中使用了基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒；所述基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒是通过将pNL4-3.Luc.R-E-骨架质粒与S蛋白或S蛋白突变体表达质粒共转染来制备的；共转染的对象为293T细胞；
所述方法中将假病毒与待评价的抗体共同孵育后加至作为靶细胞的稳定表达hACE2受体的T-REx293细胞；所述靶细胞以跨膜丝氨酸蛋白酶瞬时转染。
2.一种假病毒在评价新冠病毒疫苗效果中的应用，其特征在于，所述假病毒是基于HIV骨架并表达新冠病毒包膜蛋白突变体；所述基于HIV骨架的表达新冠病毒包膜蛋白突变体的假病毒是通过将pNL4-3.Luc.R-E-骨架质粒与S蛋白或S蛋白突变体表达质粒共转染来制备的；共转染的对象为293T细胞；所述应用中将假病毒与使用待评价的疫苗后所产生的抗体共同孵育后加至作为靶细胞的稳定表达hACE2受体的T-REx293细胞；所述靶细胞以跨膜丝氨酸蛋白酶瞬时转染。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110989583.5A CN113820495B (zh) | 2021-08-26 | 2021-08-26 | 新冠病毒包膜蛋白中和抗体活性评价方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110989583.5A CN113820495B (zh) | 2021-08-26 | 2021-08-26 | 新冠病毒包膜蛋白中和抗体活性评价方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113820495A CN113820495A (zh) | 2021-12-21 |
| CN113820495B true CN113820495B (zh) | 2024-01-26 |
Family
ID=78923411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110989583.5A Active CN113820495B (zh) | 2021-08-26 | 2021-08-26 | 新冠病毒包膜蛋白中和抗体活性评价方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113820495B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112076316A (zh) * | 2020-09-21 | 2020-12-15 | 中国人民解放军军事科学院军事医学研究院 | 一种双抗体组合物及在制备covid-19治疗药物中的应用 |
| CN112125973A (zh) * | 2020-09-30 | 2020-12-25 | 上海市公共卫生临床中心 | 冠状病毒的特异性抗体或其抗原结合片段 |
-
2021
- 2021-08-26 CN CN202110989583.5A patent/CN113820495B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112076316A (zh) * | 2020-09-21 | 2020-12-15 | 中国人民解放军军事科学院军事医学研究院 | 一种双抗体组合物及在制备covid-19治疗药物中的应用 |
| CN112125973A (zh) * | 2020-09-30 | 2020-12-25 | 上海市公共卫生临床中心 | 冠状病毒的特异性抗体或其抗原结合片段 |
Non-Patent Citations (2)
| Title |
|---|
| Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2;Jie Hu 等;Genes & Diseases;第7卷;第551-557页 * |
| LY6E Restrict Entry of Human Coronaviruses， Including Currently Pandemic SARS-CoV-2;Xuesen Zhao 等;Journal of Virology;第94卷(第18期);1-17 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113820495A (zh) | 2021-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Vogel et al. | A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates | |
| US11612649B2 (en) | HIV pre-immunization and immunotherapy | |
| EP2964769B1 (en) | Cytomegalovirus vectors enabling control of t cell targeting | |
| US20240091340A1 (en) | Pre-immunization and immunotherapy | |
| Xi et al. | Role of novel type I interferon epsilon in viral infection and mucosal immunity | |
| JP7260170B2 (ja) | 事前の免疫化ステップのないｈｉｖ免疫療法 | |
| Wang et al. | Vaginal type-II mucosa is an inductive site for primary CD8+ T-cell mucosal immunity | |
| Willer et al. | Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus | |
| Jin et al. | Induction of potent cellular immune response in mice by hepatitis C virus NS3 protein with double‐stranded RNA | |
| De Haes et al. | Lipoplexes carrying mRNA encoding Gag protein modulate dendritic cells to stimulate HIV-specific immune responses | |
| CN113820495B (zh) | 新冠病毒包膜蛋白中和抗体活性评价方法 | |
| US20100129402A1 (en) | Double-effective vaccine vector against foot-and-mouth disease virus (fmdv), methods of preparing and using the same | |
| CN117729934A (zh) | 用于疫苗递送的非病毒dna载体 | |
| Bui et al. | Schistosoma mansoni soluble egg antigens enhance T cell responses to a newly identified HIV-1 Gag H-2b epitope | |
| RU2578160C1 (ru) | Иммунобиологическое средство и способ его использования для индукции специфического иммунитета против вируса эбола (варианты) | |
| CN113185586B (zh) | SARS-CoV-2编码蛋白来源的T细胞表位多肽及其应用 | |
| CN109172818B (zh) | 一种蛋白牛痘疫苗及其效力检测方法 | |
| US11980663B2 (en) | HIV pre-immunization and immunotherapy | |
| Wade-Evans et al. | Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge | |
| Dhalech et al. | Coxsackievirus B3 elicits a sex-specific CD8+ T cell response in female mice |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |