CN115029319A - 一种人ace2过表达细胞株的构建方法 - Google Patents
一种人ace2过表达细胞株的构建方法 Download PDFInfo
- Publication number
- CN115029319A CN115029319A CN202210625435.XA CN202210625435A CN115029319A CN 115029319 A CN115029319 A CN 115029319A CN 202210625435 A CN202210625435 A CN 202210625435A CN 115029319 A CN115029319 A CN 115029319A
- Authority
- CN
- China
- Prior art keywords
- ace2
- cell
- cells
- plasmid
- human
- 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
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 title claims abstract description 20
- 102000048657 human ACE2 Human genes 0.000 title claims abstract description 20
- 230000002018 overexpression Effects 0.000 title claims abstract description 20
- 238000010276 construction Methods 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 70
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 claims abstract description 32
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 claims abstract description 32
- 239000013612 plasmid Substances 0.000 claims abstract description 24
- 238000012216 screening Methods 0.000 claims abstract description 14
- 238000004806 packaging method and process Methods 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 9
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 7
- 239000012528 membrane Substances 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 239000012634 fragment Substances 0.000 claims description 13
- 239000013613 expression plasmid Substances 0.000 claims description 12
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 12
- 241000700605 Viruses Species 0.000 claims description 6
- 229950010131 puromycin Drugs 0.000 claims description 6
- 230000014509 gene expression Effects 0.000 claims description 5
- 241000713666 Lentivirus Species 0.000 claims description 4
- 238000012761 co-transfection Methods 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 101100433975 Homo sapiens ACE2 gene Proteins 0.000 claims description 3
- 239000002502 liposome Substances 0.000 claims description 3
- 230000004807 localization Effects 0.000 claims description 3
- 230000000007 visual effect Effects 0.000 claims description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 108091008146 restriction endonucleases Proteins 0.000 claims 3
- 238000011160 research Methods 0.000 abstract description 5
- 210000000170 cell membrane Anatomy 0.000 abstract description 3
- 239000013604 expression vector Substances 0.000 abstract 2
- 101150054399 ace2 gene Proteins 0.000 abstract 1
- 102100031673 Corneodesmosin Human genes 0.000 description 11
- 101710139375 Corneodesmosin Proteins 0.000 description 11
- 241000711573 Coronaviridae Species 0.000 description 10
- 230000000903 blocking effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 5
- 108010016626 Dipeptides Proteins 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000006037 cell lysis Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 101150116411 AGTR2 gene Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000773743 Homo sapiens Angiotensin-converting enzyme Proteins 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- ZUKPVRWZDMRIEO-VKHMYHEASA-N L-cysteinylglycine Chemical compound SC[C@H]([NH3+])C(=O)NCC([O-])=O ZUKPVRWZDMRIEO-VKHMYHEASA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000056252 human ACE Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Vascular Medicine (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种ACE2过表达细胞株的构建方法，通过给人ACE2基因前面引入膜定位信号肽构建慢病毒表达载体pCDH‑CMV‑mem‑ACE2‑EF1‑GFP，与慢病毒包装质粒共转染细胞，经过抗性筛选，最终获得稳定过表达人ACE2的细胞株。应用该方法获得的过表达细胞株将人ACE2定位表达在细胞膜上，更加符合其生物学特性，为以人ACE2为靶点的相关研究提供了一种可靠的研究模型。
Description
技术领域
本发明属于生物工程技术领域，涉及新冠病毒阻断多肽筛选，具体涉及膜定位过表达ACE2细胞株在新型冠状病毒阻断肽筛选方面的应用及其制备方法。
背景技术
由于新冠病毒的高效变异性，研制新冠病毒特异性治疗药物难度非常高。作为新冠病毒在人类细胞结合受体ACE2成为研究的热点之一，ACE2也称为ACEH，即血管紧张素转化酶2。该基因编码蛋白是SARS和HCoV-NL63人类冠状病毒S糖蛋白的功能受体，同时 ACE2与ANGI、ANGII以及AT2受体有很强的亲和力，共同调节血压、体液平衡、炎症、细胞增殖、肥大和纤维化。此外该基因可能在调节心血管和肾脏功能以及生育方面也发挥了作用。
现有研究主要着眼于已知的ACE2酶活抑制剂，但由于ACE2在人体肾素-血管紧张素系统中还承担着重要的作用，抑制ACE2酶活性可能会造成ACE2对血压影响引起副作用。因此，寻找一种新型的、对ACE2酶活性无影响的抑制剂，是新型冠状病毒特效药物研发的关键。
发明内容
本发明的目的在于提供一种人ACE2过表达细胞株的构建方法，该细胞株可在其细胞膜上表达有生物活性的ACE2蛋白，利用该细胞株上的ACE2蛋白与新型冠状病毒S蛋白间的特异性作用，可用于筛选阻断新型冠状病毒与细胞结合的拮抗剂，以解决新冠病毒变异较快，疫苗可能对变异株感染没有保护作用的问题。
本发明人ACE2过表达细胞株的构建方法如下：
1.构建ACE2表达质粒
在人ACE2基因前引入膜定位信号肽mem基因构成mem-ACE2基因片段，在该片段5’端引入XbaI酶切位点、3’端引入BamHI酶切位点，酶切后克隆入pC DH-CMV-MCS-EF1-GFP质粒中，构建成pCDH-CMV-mem-ACE2-EF1-GFP载体，即ACE2表达质粒。
所述膜定位信号肽mem基因的序列：ATGCTGTGCTGTATGAGAAGAACCA AACAGGTTGAAAAGAATGATGAGGACCAAAAGATC。
2.表达质粒及慢病毒包装质粒共转染细胞
将上述ACE2表达质粒连同慢病毒包装质粒共转染细胞，嘌呤霉素进行加压筛选，观察GFP蛋白表达情况，待视野中克隆80％以上细胞呈现荧光后收集细胞并扩增，获得人ACE2过表达细胞株。
上述构建方法中，所述共转染所用细胞为293T细胞、CHO细胞、A549细胞等中任意一种。
上述构建方法中，所述共转染采用脂质体或通过电转方式将ACE2表达质粒和慢病毒包装质粒共转染细胞。
本发明相较于现有技术具有如下的有益效果：
(1)传统的人ACE2过表达细胞均为胞内表达，无法真实模拟人ACE2与其配体蛋白之间作用过程。本发明引入膜定位信号肽，使人ACE2在细胞膜上过表达，符合其生物学特性，能真实模拟病毒或其受体感染细胞过程，更有利于筛选。
(2)本发明所采用的pCDH-CMV-MCS-EF1-GFP载体上包含GFP荧光蛋白，便于在构建、筛选阶段观察实验效果。
(3)本发明利用流式细胞术进行筛选，可批量进行操作，且更灵敏、快捷。
(4)本发明方法构建的细胞株可用于筛选阻断新冠病毒S蛋白和人ACE2的受体结合的拮抗剂，使病毒无法进入细胞机体内，从而抑制病毒的扩增和存活。
附图说明
图1是pCDH-CMV-mem-ACE2-EF1-GFP质粒测序部分结果。
图2是pCDH-CMV-mem-EF1-GFP质粒、pCDH-CMV-mem-ACE2-EF1-GFP质粒分别与慢病毒包装质粒共转染293T细胞48h的荧光显微镜图(100X)，其中左图为pCDH-CMV-mem-EF1-GFP质粒转染结果，右图为 pCDH-CMV-mem-ACE2-EF1-GFP质粒转染结果。
图3是嘌呤霉素筛选所得人ACE2过表达细胞荧光显微镜结果(100X)，其中左图为明场，右图为激发荧光。
图4是人ACE2过表达细胞株WB鉴定结果，其中c为293T细胞裂解蛋白，1 为pCDH-CMV-MCS-EF1-GFP转染293T细胞裂解蛋白，2为人ACE2过表达细胞裂解蛋白。
图5是流式筛选S蛋白阻断肽结果，其中a为人ACE2过表达细胞流式结果； b为人ACE2过表达细胞中加入FITC标记S蛋白流式结构；c为人ACE2过表达细胞中先加入二肽，后加入FITC标记S蛋白后流式结果。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述。以下实施例仅用于说明本发明而不用于限制本发明的范围，该领域的技术人员可以根据上述本发明内容对发明做出一些非本质的改进和调整。未注明具体条件的实验方法，通常按照《分子克隆实验指南》中所述常规条件，或试剂制造厂商所建议的条件实施。
实施例1
1、构建ACE2表达质粒
在人ACE2基因前引入膜定位信号肽mem构成mem-ACE2基因片段，并在该片段5’端引入XbaI酶切位点，在其3’端引入BamHI酶切位点；然后将引入酶切位点后的mem-ACE2基因片段及pCDH-CMV-MCS-EF1-GFP质粒分别用XbaI 和BamHI进行双酶切，酶切体系为50μL：10xKbuffer0.5μL、XbaI5μL、BamHI5μL、质粒或mem-ACE2序列15μL、ddH2O24.5μL，上述体系混匀后置于37℃酶切8h，然后跑0.8％的琼脂糖凝胶电泳，胶回收大片段。按照pCDH-CMV-MCS-EF1-GFP： mem-ACE2＝1:5的体积比制备连接体系，用T4连接酶4℃过夜连接后，转化大肠杆菌感受态，涂布AMP抗性LB平板，37℃培养过夜后，挑取阳性克隆扩增后，提取pCDH-CMV-mem-ACE2-EF1-GFP质粒，送交测序公司测序。测序结果无误，结果见图1。同时构建pCDH-CMV-mem-EF1-GFP质粒作为对照质粒。
2、表达质粒及慢病毒包装质粒共转染293T细胞
取培养好的293T细胞，消化计数后按照8×105/孔接种到6孔板中，轻晃使其均匀铺板，37℃、5％CO2培养箱中过夜培养。将测序正确的 pCDH-CMV-mem-ACE2-EF1-GFP质粒和对照质粒分别与慢病毒包装所需三种质粒等比例转染上述过夜培养的293T细胞，转染过程参照脂质体2000说明进行。转染完成后更换新鲜的DMEM(高糖)加10％的胎牛血清培养液，其中加入2μg/mL嘌呤霉素进行筛选培养。37℃、5％CO2培养箱中培养48h后显微镜下观察细胞荧光情况，结果如图2，视野中50％以上细胞呈现荧光，表明转染成功。继续更换含有 2μg/mL嘌呤霉素的培养液持续加压筛选培养，直至视野中克隆80％以上细胞呈现荧光即可收集细胞，结果如图3所示。将收集所得细胞用DMEM(高糖)加10％的胎牛血清培养液，添加0.8μg/mL嘌呤霉素继续扩增培养后冻存。
取上述冻存细胞，复苏至T25培养瓶中扩增培养，待铺板率达到85％以上， 0.05％胰酶消化收集细胞并计数。按照每3×106个细胞加入72μL细胞裂解液，同时按比例加入8μL10x的蛋白酶抑制剂，室温裂解30min，4℃12000rpm离心10min，收集上清冻存。利用免疫印迹(WB)鉴定上述上清中人ACE2表达情况，设置293T 细胞及pCDH-CMV-MCS-EF1-GFP转染组细胞做对照，结果显示过表达细胞中人 ACE2表达量显著高于对照组，表明细胞株构建成功，结果如图4所示。
为了证明本发明的有益效果，对上述构建的ACE2过表达细胞株进行S蛋白阻断肽筛选研究。
1、细胞准备：复苏培养ACE2过表达细胞，待细胞融合度达到80％左右即可使用。
2、探针溶解：用D-PBS溶解半胱氨酸-天冬氨酸、天冬酰胺-苯丙氨酸、丙氨酸-苏氨酸、组氨酸-苯丙氨酸和半胱氨-甘氨酸五种二肽及FITC标记的S蛋白，其中FITC标记的S蛋白购自北京百普赛斯生物科技股份有限公司(货号： SPN-C52H9)。3、细胞收集：取培养好的ACE2过表达细胞，消化离心收集细胞，用D-PBS+3％FBS调整细胞密度至1×106/mL，每个处理1mL分装。
4、分装好的细胞，300g离心5min后，弃上清，实验中依次加入上述一种二肽和FITC标记的S蛋白，分别于室温孵育30min，且每次孵育完成后用D-PBS清洗 3次后用流式细胞仪检测荧光变化情况。
5、用ACE2过表达细胞调零，加入FITC标记S蛋白的ACE2过表达细胞做对照，上流式细胞仪进行检测。发现天冬酰胺-苯丙氨酸可降低S蛋白与ACE2结合后的荧光强度，预示着该二肽可能可以阻断S蛋白与ACE2之间的结合，结果如图 5所示。
Claims (3)
1.一种人ACE2过表达细胞株的构建方法，其特征在于：
(1)构建ACE2表达质粒
在人ACE2基因前引入膜定位信号肽mem基因构成mem-ACE2基因片段，在该片段5’端引入XbaI酶切位点、3’端引入BamHI酶切位点，酶切后克隆入pCDH-CMV-MCS-EF1-GFP质粒中，构建成pCDH-CMV-mem-ACE2-EF1-GFP载体，即ACE2表达质粒；
所述膜定位信号肽mem基因的序列：ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC；
(2)表达质粒及慢病毒包装质粒共转染细胞
将上述ACE2表达质粒连同慢病毒包装质粒共转染细胞，嘌呤霉素进行加压筛选，观察GFP蛋白表达情况，待视野中克隆80％以上细胞呈现荧光后收集细胞并扩增，获得人ACE2过表达细胞株。
2.根据权利要求1所述的人ACE2过表达细胞株的构建方法，其特征在于：共转染所用细胞为293T细胞、CHO细胞、A549细胞中任意一种。
3.根据权利要求1或2所述的人ACE2过表达细胞株的构建方法，其特征在于：所述共转染采用脂质体或通过电转方式将ACE2表达质粒和慢病毒包装质粒共转染细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210625435.XA CN115029319A (zh) | 2022-06-02 | 2022-06-02 | 一种人ace2过表达细胞株的构建方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210625435.XA CN115029319A (zh) | 2022-06-02 | 2022-06-02 | 一种人ace2过表达细胞株的构建方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115029319A true CN115029319A (zh) | 2022-09-09 |
Family
ID=83122084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210625435.XA Pending CN115029319A (zh) | 2022-06-02 | 2022-06-02 | 一种人ace2过表达细胞株的构建方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115029319A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043786A (zh) * | 2017-05-11 | 2017-08-15 | 合肥知恩生物技术有限公司 | 一种分泌表达蛋白的稳定细胞株构建方法 |
| CN107557391A (zh) * | 2017-09-25 | 2018-01-09 | 山东信得科技股份有限公司 | 基于Nectin4受体的犬瘟热敏感细胞系建立方法及应用 |
| CN111269937A (zh) * | 2020-02-16 | 2020-06-12 | 王清路 | 过表达hACE2基因的巨噬细胞制剂的制备方法 |
| CN111304254A (zh) * | 2020-02-20 | 2020-06-19 | 齐鲁医药学院 | 过表达hACE2基因的中性粒细胞制剂的制备方法 |
-
2022
- 2022-06-02 CN CN202210625435.XA patent/CN115029319A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043786A (zh) * | 2017-05-11 | 2017-08-15 | 合肥知恩生物技术有限公司 | 一种分泌表达蛋白的稳定细胞株构建方法 |
| CN107557391A (zh) * | 2017-09-25 | 2018-01-09 | 山东信得科技股份有限公司 | 基于Nectin4受体的犬瘟热敏感细胞系建立方法及应用 |
| CN111269937A (zh) * | 2020-02-16 | 2020-06-12 | 王清路 | 过表达hACE2基因的巨噬细胞制剂的制备方法 |
| CN111304254A (zh) * | 2020-02-20 | 2020-06-19 | 齐鲁医药学院 | 过表达hACE2基因的中性粒细胞制剂的制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| JIANHUA WANG等: "Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling", 《PLOS ONE》, pages 1 - 11 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107828738A (zh) | 一种dna甲基转移酶缺陷型cho细胞系及其制备方法及应用 | |
| CN108588028B (zh) | 一种靶向cdkn2a的cic细胞模型及其制备方法 | |
| CN102321655B (zh) | 一种基于假attP位点整合的通用型表达载体及其构建方法和应用 | |
| CN106987559A (zh) | 一种重组chok1细胞株的构建方法及其应用 | |
| CN104130977B (zh) | 一种抗肿瘤药物筛选细胞模型及其应用 | |
| CN115161286A (zh) | 一种稳定表达非洲猪瘟病毒p30蛋白CHO细胞系及其构建方法 | |
| CN102174649B (zh) | 快速检测锌指核酸酶介导基因定点整合的方法 | |
| CN115029319A (zh) | 一种人ace2过表达细胞株的构建方法 | |
| CN113897388A (zh) | 一种新型黑猩猩腺病毒载体及其构建方法和应用 | |
| CN112592388A (zh) | 一种2a肽、双顺反子表达载体、重组蛋白表达系统及应用 | |
| Lorens et al. | Stable, stoichiometric delivery of diverse protein functions | |
| CN111088272A (zh) | 一种双启动子表达载体及其构建方法 | |
| CN108753826B (zh) | 稳定表达催产素受体人类星形胶质细胞的构建方法 | |
| CN102392047A (zh) | 一种适用于哺乳动物细胞的双顺反子表达载体及其应用 | |
| CN113698466B (zh) | Ccnd1在制备禽反转录病毒生产增强剂中的应用 | |
| CN116143946A (zh) | 一种用于免疫治疗的新型融合蛋白、基因、重组载体、宿主细胞及应用 | |
| CN101463362A (zh) | 融合表达绿色荧光蛋白的表达载体及其构建方法与应用 | |
| CN112369369B (zh) | 一种人源黑素瘤细胞a375/dr5稳转株裸鼠移植瘤模型的构建方法及其应用 | |
| CN104059141B (zh) | 一种小鼠rankl突变体及其表达载体的构建、表达及应用 | |
| CN112921053A (zh) | 一种可追踪细胞分化发育的双重诱导mCreER系统及其建立与应用 | |
| CN112779262A (zh) | 猪RagC基因的应用 | |
| CN111961689A (zh) | 一种过表达pd-l1基因的间充质干细胞株及其构建方法和应用 | |
| CN104450781A (zh) | 一种过表达ciapin1蛋白的细胞系及其制备方法和应用 | |
| CN116003624B (zh) | Sirt1融合蛋白及其用途 | |
| CN111228292B (zh) | 人tpt1/tctp基因在制备抗肿瘤药物中的应用 |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220909 |
|
| RJ01 | Rejection of invention patent application after publication |