CN110063954B - Fhpi在制备治疗猫传染性腹膜炎药物中的应用 - Google Patents
Fhpi在制备治疗猫传染性腹膜炎药物中的应用 Download PDFInfo
- Publication number
- CN110063954B CN110063954B CN201910285547.3A CN201910285547A CN110063954B CN 110063954 B CN110063954 B CN 110063954B CN 201910285547 A CN201910285547 A CN 201910285547A CN 110063954 B CN110063954 B CN 110063954B
- Authority
- CN
- China
- Prior art keywords
- fhipi
- cells
- cell
- virus
- feline
- 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.)
- Expired - Fee Related
Links
- 208000005098 feline infectious peritonitis Diseases 0.000 title claims abstract description 14
- 239000003814 drug Substances 0.000 title claims description 13
- 238000002360 preparation method Methods 0.000 title description 4
- 239000003112 inhibitor Substances 0.000 claims abstract description 4
- 241000711475 Feline infectious peritonitis virus Species 0.000 claims description 8
- QHKYPYXTTXKZST-UHFFFAOYSA-N SB-202190 Chemical compound C1=CC(O)=CC=C1C1=NC(C=2C=CC(F)=CC=2)=C(C=2C=CN=CC=2)N1 QHKYPYXTTXKZST-UHFFFAOYSA-N 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 2
- 125000001255 4-fluorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1F 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 27
- 230000000694 effects Effects 0.000 abstract description 19
- 241000725579 Feline coronavirus Species 0.000 abstract description 13
- 230000005764 inhibitory process Effects 0.000 abstract description 12
- 238000001262 western blot Methods 0.000 abstract description 6
- 241000282326 Felis catus Species 0.000 abstract description 5
- 238000010166 immunofluorescence Methods 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 2
- 230000035755 proliferation Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 57
- 239000007788 liquid Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000005406 washing Methods 0.000 description 9
- 230000010261 cell growth Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- 238000012423 maintenance Methods 0.000 description 6
- 238000010998 test method Methods 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000034217 membrane fusion Effects 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 241000711573 Coronaviridae Species 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 3
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 241000282324 Felis Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000010023 transfer printing Methods 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- MPDDTAJMJCESGV-CTUHWIOQSA-M (3r,5r)-7-[2-(4-fluorophenyl)-5-[methyl-[(1r)-1-phenylethyl]carbamoyl]-4-propan-2-ylpyrazol-3-yl]-3,5-dihydroxyheptanoate Chemical compound C1([C@@H](C)N(C)C(=O)C2=NN(C(CC[C@@H](O)C[C@@H](O)CC([O-])=O)=C2C(C)C)C=2C=CC(F)=CC=2)=CC=CC=C1 MPDDTAJMJCESGV-CTUHWIOQSA-M 0.000 description 1
- QZDDFQLIQRYMBV-UHFFFAOYSA-N 2-[3-nitro-2-(2-nitrophenyl)-4-oxochromen-8-yl]acetic acid Chemical compound OC(=O)CC1=CC=CC(C(C=2[N+]([O-])=O)=O)=C1OC=2C1=CC=CC=C1[N+]([O-])=O QZDDFQLIQRYMBV-UHFFFAOYSA-N 0.000 description 1
- YKRNPHOBDOUQTG-UHFFFAOYSA-N 2-[4-[4-(4-carbamimidoylphenyl)piperazin-1-yl]piperidin-1-yl]acetic acid;trihydrochloride Chemical compound Cl.Cl.Cl.C1=CC(C(=N)N)=CC=C1N1CCN(C2CCN(CC(O)=O)CC2)CC1 YKRNPHOBDOUQTG-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- 108010049990 CD13 Antigens Proteins 0.000 description 1
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 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 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 206010034674 peritonitis Diseases 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了FHPI在治疗猫传染性腹膜炎中的新用途。FHPI对猫正常细胞的毒性作用很小,而对猫冠状病毒具有明显的抑制作用,能明显降低病毒在细胞中的毒价;间接免疫荧光和Western Blot的测定结果表明,FHPI能抑制猫冠状病毒在细胞中的增殖,且具有剂量依赖性。因此,FHPI具有抗猫冠状病毒的活性,可用于制备猫冠状病毒抑制剂,用于治疗猫传染性腹膜炎,具有疗效好,安全性高等优点。
Description
技术领域
本发明属于制药领域,具体涉及FHPI在制备治疗猫传染性腹膜炎的药物中的新用途。
背景技术
猫传染性腹膜炎(FIP)是由致病性冠状病毒引起的一种慢性、进行性、致死性疾病,以腹膜炎、胸腔大量积水为主要特征,病猫致死率较高,大多数药物对其没有治疗效果,只能延缓其生命。
目前对于猫传染性腹膜炎的治疗主要是采用抗病毒药、免疫调节剂和免疫抑制剂进行对症治疗,但是这些药物都不能降低FIP的高死亡率,仅能短暂延缓疾病进程。因此亟待发现一种新的治疗猫传染性腹膜炎的药物。
FHPI是一种p38MAPK抑制剂,靶向作用于p38α/β,能激活p38(MAPK)途径导致外周血单核细胞(PBMC)产生促炎细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素-1β,而TNF-α能够诱导猫T细胞凋亡。此外,TNF-α会促进FCoV受体氨肽酶N(APN)的表达,导致靶细胞更容易受到病毒感染,进一步加剧疾病。FHPI通过抑制p38MAPK来达到抗病毒效果。FHPI的化学名称为4-[4-(4-氟苯基)-5-吡啶-4-基-1,3-二氢-咪唑-2-亚基]环己-2,5-二烯-1-酮,分子式为C20H14N3OF,分子量为331.34,结构式如下:
经查阅相关文献,未见FHPI在治疗猫传染性腹膜炎方面的相关报道。
发明内容
本发明的目的是提供FHPI在制备治疗猫传染性腹膜炎药物中的新用途。
体外细胞试验结果表明:FHPI对猫正常细胞的毒性作用很小,而对猫冠状病毒具有明显的抑制作用,能明显降低病毒在细胞中的毒价;间接免疫荧光检测和Western Blot的测定结果表明,FHPI能抑制猫冠状病毒在细胞中的增殖,且具有剂量依赖性。
因此,FHPI具有抗猫冠状病毒的活性,可用作猫冠状病毒抑制剂,用于治疗由猫冠状病毒引起的各种疾病,如猫传染性腹膜炎等,具有疗效好,安全性高等优点。
附图说明
图1是FHPI对猫肾细胞的毒性试验结果。
图2是FHPI在细胞水平对猫冠状病毒的抑制显微图片。
图3是FHPI抗猫冠状病毒活性的EC50测定曲线。
图4是FHPI抗猫冠状病毒活性的TCID50测定结果。
图5是FHPI抗猫冠状病毒活性的间接免疫荧光测定结果。
图6是FHPI抗猫冠状病毒活性的Western Blot鉴定结果。
具体实施方式
下面结合实施例对本发明进行详细说明。需要说明的是,本发明的实施例仅限于对本发明进行说明,而没有限制作用。实施例中所涉及的有关试验方法和其它各种实验操作,均为本领域的常规技术,文中没有特别说明的部分,本领域的普通技术人员可以参照本发明申请日之前的各种常用工具书、科技文献或相关的说明书、手册等予以实施。
实施例1FHPI对细胞的毒性试验
试验方法:
1)取生长状态良好的CRFK细胞(猫肾细胞)进行消化传代,用细胞生长液调整细胞密度为1×105/mL接种96孔板,100μL/孔,放置于37℃、5%CO2培养箱培养16h;
2)16h后,弃去孔中培养基,1×PBS洗三次,甩干后用细胞维持液对FHPI进行2倍倍比稀释,使FHPI终浓度为200μM、100μM、50μM、25μM、12.5μM、6.25μM、3.125μM,同时设置细胞对照;
试验结果:
结果见图1。测定细胞存活率能反应FHPI对CRFK细胞的毒性作用,从图中可以看出,FHPI对CRFK细胞的毒性作用较小,在低浓度药物作用下(<50μM)可以促进细胞生长,增加细胞活力,其半数细胞毒性浓度(CC50)大于200μM。
实施例2FHPI在细胞水平对病毒的抑制效果
试验方法:
1)取生长状态良好的CRFK细胞进行消化传代,用细胞维持液调整细胞密度为1×105/mL接种于96孔板,100μL/孔,放置于37℃、5%CO2培养箱培养16h;
2)16h后,弃去孔中培养基,1×PBS洗三次,甩干后用细胞生长液对FHPI进行2倍倍比稀释,同时每孔加入0.01MOI猫冠状病毒(FIPV)。使FHPI终浓度为100μM、50μM、25μM、12.5μM,同时设置细胞对照,病毒对照;
3)待病毒对照病变70%时,显微镜观察化合物与病毒共同作用后的细胞状态变化并拍照保存。
试验结果:
结果见图2。从图中显微镜图片可以观察到正常的CRFK细胞生长状态良好,细胞形态完整,细胞边界清晰;而用病毒感染的细胞出现大片膜融合,核聚集现象,无完整的细胞形态,病变很明显;但是加入FHPI处理细胞后,细胞生长状态良好,无明显病变。由此可见FHPI在细胞水平对FIPV有一定的抑制效果。
实施例3FHPI抗病毒活性(EC50)试验
试验方法:
1)取生长状态良好的CRFK细胞进行消化传代,用细胞生长液调整细胞密度为1×105/mL接种于96孔板,100μL/孔,放置于37℃、5%CO2培养箱培养16h;
2)16h后,弃去孔中培养基,1×PBS洗三次,甩干后每组第一横排加入100μL细胞维持液,第二、三、四、五、六、七排加50μL细胞维持液;
3)在盖子上做好标记,按标记顺序每组第一横排加入FHPI,进行2倍倍比稀释,用排枪轻轻混匀十下,吸出50μL加入第二纵列,依次类推,最后混匀弃掉50μL,使FHPI终浓度为100μM、50μM、25μM、12.5μM、6.25μM、3.125μM、1.5625μM。同时每孔加入0.01MOI病毒液。设置细胞对照和病毒对照;
试验结果:
结果见图3。通过测定细胞存活率来计算药物对病毒的抑制率能反应出FHPI对FIPV的抑制效果,从图中可以看出,FHPI对FIPV的抑制效果呈剂量依耐性。其半数最大效应浓度(EC50)为:6.652μM。
实施例4FHPI抗病毒活性(TCID50)的测定
试验方法:
1)将CRFK细胞进行消化传代,用细胞生长液调整细胞密度为1×105/mL接种12孔板,1mL/孔,放置于37℃、5%CO2培养箱培养16h;
2)16h后,稀释6个浓度的FHPI,分别为50μM、25μM、12.5μM、6.25μM、3.125μM、1.5625μM,做好标记。准备EP管,加入细胞维持液与稀释好的FHPI,使12孔板的终浓度为上述6个浓度,振荡器上混匀至少5s;
3)稀释完成后,取出12孔板,弃去12孔板孔中培养基,用1×PBS清洗三次,甩干后加入已经稀释好的FHPI。置于37℃、5%CO2培养箱孵育1h;
4)1h后,取出12孔板,每孔接种0.01MOI的病毒,轻晃12孔板混匀,每块12孔板设置病毒对照与细胞对照。于37℃、5%CO2培养箱培养;
5)约18h后,病毒对照出现70%病变,将12孔板转移至-80℃超低温冰箱冻融一次;
6)收集每孔液体至EP管中,4℃4000rpm离心10min后收集上清测毒价。
试验结果:
结果见图4。整体都是随着FHPI浓度增加,病毒抑制效果越明显,呈剂量依耐性。在FHPI浓度为50μM时,抑制效果最明显。FHPI在浓度为50μM、25μM、12.5μM、6.25μM、3.125μM、1.5625μM、0μM时的TCID50分别为:10-3.30/mL、10-3.45/mL、10-3.97/mL、10-6.68/mL、10-6.86/mL、10-7.05/mL、10-7.22/mL。FHPI在浓度为50μM时可以降低3.92个滴度,浓度为25μM时可以降低3.77个滴度,浓度为12.5μM时可以降低3.25个滴度,浓度为6.25μM时可以降低0.54个滴度,浓度为3.125μM时可以降低0.36个滴度。
实施例5FHPI对病毒活性间接免疫荧光检测
试验方法:
1)铺细胞:CRFK细胞长至90%时进行传代,用细胞生长液将细胞密度调节为1×105个/mL接种96孔板,100μL/孔,放置于37℃、5%CO2培养箱培养16h。
2)病毒感染:16h后,待细胞长满单层,取出96孔板用1×PBS洗两遍,换成细胞维持液,加入化合物进行2倍倍比稀释,加入等体积0.01MOI病毒液,使化合物终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM,1.5625μM,设置病毒对照组和细胞对照组,做好标记,放置于37℃、5%CO2细胞培养箱继续培养。
3)固定细胞:待病毒对照组细胞出现70%病变(能观察到膜融合且无大片脱落),弃掉孔中培养基,用1×PBS清洗2遍,每孔加入100μL4%多聚甲醛室温固定细胞30min;弃掉多聚甲醛,用1×PBS清洗3遍,每孔加入100μL1%Triton100室温通透10min。
4)封闭:将固定好的板子用1×PBS洗3遍,每孔加入100μL5%BSA封闭,37℃封闭30min(可4℃过夜封闭);
5)加一抗:弃去封闭液,1×PBS洗3遍,每孔加入100μL1:1000稀释的一抗(N蛋白兔多克隆抗体),37℃孵育1h;
6)加二抗:弃掉一抗,1×PBS洗3遍,每孔加入100μL1:500稀释的二抗(FITC标记的羊抗兔IgG),37℃孵育1h(注意避光操作);
7)弃掉二抗(注意避光操作,1×PBS洗3遍,每孔加入50μL1×PBS,荧光显微镜下观察并拍照保存。
试验结果:
结果见图5。整体都是随着FHPI浓度增加,病毒抑制效果越明显,呈剂量依耐性。在FHPI浓度为50μM时,抑制效果最明显,基本看不见明显荧光,随着药物浓度降低,荧光越明显,能检测到的FIPV越多,在1.5625μM时,能观察到大面积的CRFK细胞感染FIPV后出现的膜融合荧光病变。
实施例6FHPI对病毒活性Western Blot的测定
试验方法:
1)铺细胞:CRFK细胞长至90%时进行传代,用细胞生长液将细胞密度调节为1×105个/mL接种6孔板,2mL/每孔,放置于37℃、5%CO2培养箱培养16h。
2)感染:16h后,待细胞密度达到85%,1×PBS润洗两次,加入化合物进行2倍倍比稀释,同时加入0.01MOI病毒液,使化合物终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM,1.5625μM,设置病毒对照组和细胞对照组,做好标记,放置于37℃、5%CO2细胞培养箱继续培养。
3)收集细胞(全程冰上操作):病毒感染24h后待细胞病变约70%(能观察到膜融合且无大片脱落),弃去孔中培养基,用预冷的1×PBS润洗一次,加入500μL预冷1×PBS,刮下细胞,收集于EP管中。4℃4000r/min离心10min。
4)裂解细胞:离心弃去上清,在沉淀中加入300μL细胞裂解液,重悬后放置4℃摇床充分裂解30min,4℃4000r/min离心20min。
5)样品制备:收集上清,加入5×蛋白缓冲液,沸水中煮10min,-20℃静置保存。
6)SDS-PAGE:配制12%分离胶和5%浓缩胶,加入1×甘氨酸电泳缓冲液,上样,80v恒压1h,120V恒压1h。
7)Western Blot湿转:采用湿法转印PVDF膜,根据蛋白大小,120V湿转40min;
8)封闭:用镊子将膜放入一个干净的平皿内,加入适量的5%脱脂牛奶封闭液,置于室温摇床2h;
9)加一抗:弃去封闭液,加入1:2000稀释的一抗(N蛋白兔多克隆抗体),置于室温摇床3h,用TBST将膜清洗三遍,每次15min;
10)加二抗:加入1:5000稀释的二抗(HRP标记的羊抗兔IgG),室温摇床1h,TBST洗三遍,每次15min;
11)显色:将A液和B液等体积混匀,于显色仪器中显色,拍照保存。
12)显色拍照后用TBST将膜清洗三遍,每次15min。同样方法孵育GAPDH一抗与羊抗鼠二抗,显色后拍照保存。
试验结果:
结果见图6。整体都是随着FHPI浓度增加,病毒抑制效果越明显,呈剂量依耐性。在FHPI浓度为50μM时,抑制效果最明显,Western Blot检测灰度值最浅,随着药物浓度降低,灰度值越明显,能检测到的FIPV越多。
Claims (2)
1.FHPI在制备猫传染性腹膜炎病毒抑制剂中的应用,所述FHPI的化学名称为4-[4-(4-氟苯基)-5-吡啶-4-基-1,3-二氢-咪唑-2-亚基]环己-2,5-二烯-1-酮。
2.权利要求1中所述的FHPI在制备治疗猫传染性腹膜炎药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910285547.3A CN110063954B (zh) | 2019-04-10 | 2019-04-10 | Fhpi在制备治疗猫传染性腹膜炎药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910285547.3A CN110063954B (zh) | 2019-04-10 | 2019-04-10 | Fhpi在制备治疗猫传染性腹膜炎药物中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110063954A CN110063954A (zh) | 2019-07-30 |
CN110063954B true CN110063954B (zh) | 2021-08-06 |
Family
ID=67367436
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910285547.3A Expired - Fee Related CN110063954B (zh) | 2019-04-10 | 2019-04-10 | Fhpi在制备治疗猫传染性腹膜炎药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110063954B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117338775A (zh) * | 2020-06-16 | 2024-01-05 | 格格巫(珠海)生物科技有限公司 | 一种化合物在预防和/或治疗动物的病原体感染中的应用 |
CN113679716B (zh) * | 2021-10-13 | 2024-03-26 | 史大永 | 溴酚-吡唑啉化合物在治疗猫冠状病毒疾病中的应用 |
CN114921424B (zh) * | 2022-06-10 | 2023-07-07 | 华中农业大学 | 一种重组猫传染性腹膜炎病毒及其应用 |
CN115105502B (zh) * | 2022-06-27 | 2023-08-22 | 陕西秦岭七药协同创新中心有限公司 | 一种含千金藤属植物生物碱的化合物在制备猫传染性腹膜炎药物中的应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2949004A1 (en) * | 2014-05-16 | 2015-11-19 | Atriva Therapeutics Gmbh | Novel anti-infective strategy against influenza virus and s. aureus coinfections |
-
2019
- 2019-04-10 CN CN201910285547.3A patent/CN110063954B/zh not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN110063954A (zh) | 2019-07-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110063954B (zh) | Fhpi在制备治疗猫传染性腹膜炎药物中的应用 | |
Goldsmith et al. | Modern uses of electron microscopy for detection of viruses | |
Hoorn et al. | On the growth of certain “newer” respiratory viruses in organ cultures | |
Sieg et al. | A new genotype of feline morbillivirus infects primary cells of the lung, kidney, brain and peripheral blood | |
Hao et al. | Canine circovirus suppresses the type I interferon response and protein expression but promotes CPV-2 replication | |
CN113813269B (zh) | Perifosine在制备抗汉滩病毒药物中的应用 | |
Qian et al. | Isolation and identification of porcine deltacoronavirus and alteration of immunoglobulin transport receptors in the intestinal mucosa of PDCoV-infected piglets | |
JPH09176043A (ja) | 魚類用のイリドウイルス感染症ワクチンと診断剤並びにこれ等の製法 | |
CN104560865A (zh) | 抗a亚群禽白血病病毒的细胞系及其构建方法与应用 | |
CN112717128B (zh) | 一种预防手足口病的联合疫苗及其制备方法和应用 | |
CN117695263A (zh) | 异甘草素在制备治疗猫传染性腹膜炎的药物中的应用 | |
AU2020102162A4 (en) | Pig tonsil cell line susceptible to jev and construction method thereof | |
CN111973600B (zh) | 霉酚酸或其衍生物在制备犬瘟热病毒抑制剂中的用途 | |
CN103864931B (zh) | 一种伪狂犬病标准阳性血清的制备及其冻干保存方法 | |
CN105861445A (zh) | 一株抗k亚群禽白血病病毒的细胞系及其应用 | |
CN109364083B (zh) | 盐酸考尼伐坦在制备治疗猫传染性腹膜炎药物中的用途 | |
Fattal et al. | Enterovirus types in Israel sewage | |
Vissani et al. | Equine coital exanthema: New insights on the knowledge and leading perspectives for treatment and prevention | |
CN116615234A (zh) | 一种预防手足口病的联合疫苗及其制备方法和应用 | |
Li et al. | Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish | |
CN106085965B (zh) | 一种鸭坦布苏病毒、由该病毒制备的疫苗及其制备方法 | |
CN115414349B (zh) | 芹菜素在制备治疗猫鼻支气管炎药物中的用途 | |
CN114984006B (zh) | 3,3`-二吲哚甲烷在制备治疗猫传染性腹膜炎的药物中的应用 | |
CN105349498B (zh) | 一种优化制备灭活疫苗和/或减毒活疫苗的方法 | |
CN112791179B (zh) | 一种预防手足口病的联合疫苗及其制备方法和应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210806 |
|
CF01 | Termination of patent right due to non-payment of annual fee |