CN105974119A - G种肠道病毒直接免疫荧光试剂及其试剂盒 - Google Patents
G种肠道病毒直接免疫荧光试剂及其试剂盒 Download PDFInfo
- Publication number
- CN105974119A CN105974119A CN201610444162.3A CN201610444162A CN105974119A CN 105974119 A CN105974119 A CN 105974119A CN 201610444162 A CN201610444162 A CN 201610444162A CN 105974119 A CN105974119 A CN 105974119A
- Authority
- CN
- China
- Prior art keywords
- enterovirus
- reagent
- immunofluorescent
- monoclonal antibody
- immunofluorescent reagent
- 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.)
- Granted
Links
- 241000709661 Enterovirus Species 0.000 title claims abstract description 43
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 42
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 15
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 238000001514 detection method Methods 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 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 claims description 8
- 238000002372 labelling Methods 0.000 claims description 8
- 238000013096 assay test Methods 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 claims 1
- 230000003612 virological effect Effects 0.000 claims 1
- 101710197658 Capsid protein VP1 Proteins 0.000 abstract description 20
- 101710132601 Capsid protein Proteins 0.000 abstract description 19
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 abstract description 19
- 101710108545 Viral protein 1 Proteins 0.000 abstract description 19
- 238000010166 immunofluorescence Methods 0.000 abstract description 15
- 238000000034 method Methods 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 9
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 206010014909 Enterovirus infection Diseases 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 244000052769 pathogen Species 0.000 abstract description 2
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- 230000004807 localization Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 22
- 238000012360 testing method Methods 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000009182 swimming Effects 0.000 description 9
- 238000000746 purification Methods 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000012797 qualification Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000701922 Bovine parvovirus Species 0.000 description 3
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000709691 Enterovirus E Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 206010068676 Pneumoretroperitoneum Diseases 0.000 description 1
- 208000005727 Retropneumoperitoneum Diseases 0.000 description 1
- 241001523574 Sheep enterovirus Species 0.000 description 1
- 101710106388 Structural protein VP1 Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 101150024766 VP1 gene Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000726445 Viroids Species 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 239000012224 working solution Substances 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了检测G种肠道病毒的免疫荧光试剂及其检测试剂盒,是用荧光染料也可标记的VP1单克隆抗体,所述的VP1单克隆抗体,是利用G种肠道病毒的VP1蛋白,采用杂交瘤单抗制备技术获得;所述的VP1蛋白的氨基酸序列如序列表SEQ ID NO.2所示。既可用于临床快速诊断G种肠道病毒感染,也可用于G种肠道病毒抗原在组织中的定位与鉴定;直接免疫荧光检测时间短,40分钟内即可报告结果;特异性强,仅与G种肠道病毒反应;重复性好,准确性高,与间接免疫荧光方法的符合率为90%以上,可用于G种肠道病毒感染的快速诊断。
Description
技术领域
本发明属生物技术领域,具体涉及检测G种肠道病毒直接免疫荧光试剂及其试剂盒,适于G种肠道病毒感染的诊断及组织或细胞中羊肠道病毒抗原的定位与鉴定。
背景技术
羊G种肠道病毒(CEV),属于小RNA病毒科肠道病毒属。羊肠道病毒感染是目前国内外新发的一种临床上以消化道和呼吸道为主要特征的疫病,给养羊业带来严重威胁。申请人于2014年从吉林多地爆发的一种临床上以严重腹泻,高发病率和高致死率为主要特征的不明疫病中的山羊体内分离到G种肠道病毒CEV-JL14,经过序列测定与分析发现,所分离的毒株是一种新的肠道病毒。目前,针对该类病毒或该类疫病尚没有特异性的检测与诊断方法。
发明内容
本发明的目的是为解决羊G种肠道病毒尚没有特异性的检测与诊断方法提供一种检测G种肠道病毒的直接免疫荧光检测荧光试剂及其试剂盒。
检测G种肠道病毒的免疫荧光试剂,是用FITC标记(但不仅限于FITC,其他荧光染料也可)的VP1单克隆抗体,所述的VP1单克隆抗体,是利用G种肠道病毒的VP1蛋白,采用杂交瘤单抗制备技术获得;所述的VP1蛋白的氨基酸序列如序列表SEQ ID NO.2所示;
所述的VP1蛋白是由碱基序列如序列表SEQ ID NO.1所示的G种肠道病毒基因VP1表达的。所述的G种肠道病毒基因VP1,可采用PCR技术,以G种肠道病毒基因组cDNA为模板扩增获得,也可人工合成;
G种肠道病毒直接免疫荧光检测试剂盒,免疫荧光试剂为上述检测G种肠道病毒的免疫荧光试剂。
本发明提供了检测G种肠道病毒的免疫荧光试剂及其检测试剂盒,是应用本发明的检测G种肠道病毒的免疫荧光试剂建立的直接免疫荧光检测方法,既可用于临床快速诊断G种肠道病毒感染,也可用于G种肠道病毒抗原在组织中的定位与鉴定。直接免疫荧光检测时间短,40分钟内即可报告结果;特异性强,仅与G种肠道病毒反应;重复性好,准确性高,与间接免疫荧光方法的符合率为90%以上,可用于G种肠道病毒感染的快速诊断。
本发明的优点
1、应用本发明试剂建立的直接免疫荧光方法,可用于临床快速诊断G种肠道病毒感染,也可用于G种肠道病毒抗原在组织细胞中的定位与鉴定;
2、直接免疫荧光方法特异性强、灵敏度高、具有很好的重复性。40分钟内报告结果,可用于G种肠道病毒的快速诊断;
3、具有生物安全性,试剂盒所用的抗G种肠道病毒VP1的单克隆抗体为原核载体表达的重组蛋白诱导BALB/c小鼠产生,不含活的肠道病毒,因此不存在散毒危险;
4、敏感性高,制备单克隆抗体具有很高的免疫效价,从而确保异硫氰酸荧光素(FITC)标记后试剂的敏感性;
5、特异性强,制备的免疫荧光试剂与其他病原体无交叉反应,只检出G种肠道病毒,具有很高特异性;
6、稳定性好,荧光试剂4℃保存6个月,检测结果与常规方法无明显差异,具有很好的稳定性。
附图说明
图1 pGEX-4T-1-VP1重组质粒酶切鉴定结果(泳道M:DNA分子marker;泳道1:pGEX-4T-1-VP1 EcoRI/XhoI双酶切;泳道2:pGEX-4T-1-VP1 EcoRI单酶切);
图2重组VP1蛋白的SDS-PAGE检测结果(泳道M:蛋白质分子量marker;泳道1 :pGEX-4T-1-VP1重组菌未诱导总蛋白;泳道2:pGEX-4T-1-VP1重组菌诱导后总蛋白;泳道3:纯化后的重组VP1蛋白);
图3杂交瘤细胞染色体计数;
图4. 直接免疫荧光检测结果。
具体实施方式
实施例1 G种肠道病毒结构蛋白VP1原核表达重组质粒的构建
1、引物设计
根据目标序列的碱基组成,分别设计引物扩增VP1基因,上下游分别含有EcoRI、XhoI酶切位点。引物序列如下:
上游引物:5' TTGAATTCCAGGCTGGGTATGTGACT 3' (EcoRI)
下游引物:5' TCTCGAGTTAATGAAAATCACTGAGGGGTT 3' (XhoI)
2、基因扩增
常规Trizol法提取G种肠道病毒的基因组RNA,采用市售常规反转录试剂盒,按照说明操作获得cDNA,并以其为模板扩增VP1基因,PCR反应体系:
PCR运行条件:94℃预变性3min;94℃变性1.5min、54℃退火40sec、72℃延伸40sec,共35个循环;72℃再延伸6min。反应结束后,取1μL进行0.8%琼脂糖凝胶电泳检测。
3、目标基因与pGEX-4T-1的连接、转化
采用分子生物学领域中的常规酶切、连接等技术,构建重组质粒pGEX-4T-1-VP1,并采用CaCl2法将重组质粒转化大肠杆菌DH5α感受态中。利用含100 µg/mL氨苄霉素的LB培养平板筛选阳性克隆,并进行常规增菌培养,收获菌体,提取质粒,进行EcoRI/XhoI双酶切鉴定,如图1所示。
实施例2 VP1重组蛋白的表达及纯化
将鉴定的阳性重组质粒pGEX-4T-1-VP1转化大肠杆菌BL21(DE3) 感受态细胞后,涂板,37℃培养过夜,挑取单个菌落接种到3 mL含有100 µg/mL氨苄霉素的LB液体培养基中培养过夜,然后取1 mL上述培养物接种到200 mL含有100 µg/mL氨苄霉素的LB液体培养基中37℃振摇培养至对数生长期(OD600=0.6),加入IPTG(终浓度1 mmol/L),20℃诱导培养3 h,经SDS-PAGE检测,获得了重组目标蛋白VP1,如图2所示(泳道2)。离心沉淀菌体, 超声破碎后以1.5M尿素洗涤3次后获得高纯度的重组蛋白,如图2所示(泳道3)。
实施例3 抗G种肠道病毒VP1蛋白单克隆抗体的制备
1、动物免疫:选择6-8周龄健康BALB/c小鼠,以弗氏完全佐剂乳化纯化的VP1蛋白,每只小鼠腹腔注射100µg,14d后以弗氏不完全佐剂乳化蛋白腹腔注射100µg,最后一次加强免疫时直接腹腔注射100µg纯化的蛋白,融合前3~4d尾静脉注射50µg纯化的蛋白。
2、细胞融合:取免疫小鼠的脾细胞与SP2/0混合于融合管内, 以300×g离心10min, 弃去上清, 振荡细胞, 使两种细胞尽量混合均匀, 然后60s内缓慢滴加预热的PEG-4000溶液,再缓慢加入无血清的1640培养基终止融合, 静置后再以1 000 r/min离心10min , 弃去上清后加入HAT培养基,使细胞混匀悬浮,于96孔培养板中培养,第14d开始换液,并检测杂交瘤细胞。
3、杂交瘤细胞克隆的筛选:采用间接ELISA方法,即用纯化的VP1蛋白作为包被抗原,包被反应板,加入杂交瘤细胞的培养上清进行ELISA检测;对ELISA阳性的杂交瘤细胞进行克隆,直至所有孔都为阳性。通过3次细胞克隆,获得20株杂交瘤细胞,其中用人工合成VP1基因表达的VP1蛋白免疫,取免疫小鼠的脾细胞进行杂交,获得杂交瘤细胞12株;PCR方法获得的基因,获得杂交瘤细胞8株;
按上述方法,经多次重复,均获得了筛选到了多株杂交瘤。
4、单克隆抗体制备:取高压灭菌的石蜡油0.5 mL,注射到小鼠腹腔,7d后腹腔注入106个杂交瘤细胞,一周后抽取腹水,置37℃ 24 h后4℃过夜,然后离心腹水,上清56℃灭活30 min备用。
5、单克隆抗体纯化:用4倍体积醋酸缓冲液稀释腹水,调至pH4.5,缓慢滴加3.3 %的正辛酸,搅拌30 min,离心取上清,加入1/10体积的10倍PBS,调至pH7.4,4℃预冷后用45%饱和硫酸铵沉淀,离心后将沉淀以PBS溶解后移入透析袋,在50倍体积PBS中透析,透析期间每6小时换液1次,透析结束后,以紫外分光光度计测定蛋白浓度并分装冻存。
6、单克隆抗体特性鉴定
(1)腹水效价确定。以间接ELISA方法测定。细胞培养上清与腹水以10×进行倍比稀释,同时以SP2/0 细胞培养上清和未接肿瘤细胞健康小鼠的腹水作为对照。检测确定出单抗的腹水效价为6.4×105。
(2)抗VP1蛋白单克隆抗体IgG亚类鉴定。按小鼠mAbIgG亚类检测试剂盒说明书进行。具体方法是将纯化的每株单克隆抗体1000倍稀释后包被酶标板,每孔100 µL,37℃孵育1 h,洗涤后每孔加入1000倍稀释的抗体亚类试剂100 µL,37℃孵育1 h,洗涤3遍后,加入羊抗小鼠IgG酶标二抗, 37℃孵育1 h,确定单克隆抗体的亚类为IgG1。
(3)杂交瘤细胞株染色体的分析采用秋水仙素法对杂交瘤细胞株进行染色体分析,结果显示杂交瘤细胞的染色体数目为86-90条。
(4)抗VP1单克隆抗体特异性鉴定。将所获单克隆抗体分别与口蹄疫病毒(FMDV)、牛病毒性腹泻病毒(BVDV)、牛细小病毒(BPV)进行间接ELISA,结果显示,制备的单克隆抗体与其它病毒病均无交叉反应性,鉴定结果见表1。
实施例4 免疫荧光抗体的制备
1、抗体稀释:将VP1单克隆抗体以碳酸盐缓冲液(0.025 M pH9.2 CBS)稀释为蛋白浓度为10 mg/mL,装入透析袋中备用。
2、荧光素溶液制备:称取异硫氰酸荧光素(FITC),用0.025 M pH9.2 CBS配制成0.1 mg/mL,磁力搅拌避光溶解。
3、抗体标记:将抗体和荧光色素混合,加入到透析袋4℃避光标记24 h。
4、荧光标记抗体的纯化:取出透析袋中的标记抗体,1000 r/min离心3 min,取上清液过SephandxG-50层析柱,用灭菌0.01 M pH7.2 PBS洗脱,除去游离的荧光素和盐;分管收集第一峰,适当浓缩,测定其工作浓度后分装,保存于-20℃备用,即为牛肠道病毒免疫荧光诊断试剂。
实施例5直接免疫荧光试验方法的建立
1、CEV感染细胞的制备:将G种肠道病毒(CEV)接种于已长成单层的96孔RAW264.7细胞培养板,同时设正常RAW264.7细胞为阴性对照;于37℃ 5% CO2条件下培养至细胞病变达30% 左右时,弃上清液,以无菌PBS或生理盐水洗涤1,每孔加入冷甲醇100 µL,4℃固定30min,弃去固定液同前洗涤1次,-20℃冻存备用。
2、直接免疫荧光试验操作方法:取CEV感染细胞板,加入适量经1 % BSA- PBS稀释的上述免疫荧光试剂工作液,置于湿盒内,37℃感作30 min,用PBS或生理盐水洗涤3次后,每孔加30 µL抗荧光衰减封片剂(50 %甘油-PBS),置倒置荧光显微镜下观察,以出现亮绿色荧光病灶判定阳性结果,拍照保存。同时设未接种病毒的正常细胞孔作为阴性对照。
实施例6免疫荧光试剂特性鉴定
1、荧光试剂工作浓度的确定:取实施例5中制备的感染细胞板和阴性细胞板作为对照,每孔分别加入预先经1 % BSA-PBS系列稀释的免疫荧光试剂,进行直接免疫荧光试验,以出现清晰、明亮、特异性荧光的稀释倍数为试剂的最大稀释倍数。结果显示,荧光试剂的最高稀释倍数为1:1000。为保证试剂的敏感性和荧光强度,确定工作浓度为1:500。
2、荧光试剂的特异性检测:将荧光抗体适当稀释后,分别与FMDV、BVDV和BPV感染的细胞感作,同时设阴性对照,每孔加1:500稀释的荧光试剂30 µL,进行直接免疫荧光试验试验。结果显示,在荧光显微镜下仅CEV感染细胞孔出现亮绿色荧光病灶,呈阳性反应,直接免疫荧光试验的荧光特性与IFA相同;而其它病毒感染细胞及正常RAW264.7细胞均无交叉反应。表明制备的免疫荧光试剂特异性好,可用于CEV感染细胞的鉴定,见表2。
3、免疫荧光试剂的重复性和稳定性检测:在96孔细胞培养板中进行,将抗VP1荧光试剂分装后分别置-20℃保存1、2、3、4、5和6个月后,对CEV感染细胞进行直接免疫荧光试验试验,测定试剂保存期和批内批间重复性。结果显示,在-20℃保存至6个月荧光效价不变,因此其保存期为6个月;同时,批内和批间重复性较好,质量稳定。
实施例7 其他颜色荧光染料标记
采用红色荧光料PE,按照实施例4操作,标记抗G种病毒VP1单克隆抗体获得的免疫荧光试剂,其检测效果与FITC标记的免疫荧光试剂相似。
<110> 吉林大学
<120> G种肠道病毒直接免疫荧光试剂及其试剂盒
<160> 2
<210> 1
<211> 677
<212> DNA
<213> 人工
<400> 1
caggctgggt atgtgactgg ttggtaccag accaacatgg tgatccctcc cgagtttccc 60
cagaccgcta atatcatctg tttagtggca gcacaaccaa atttttccct gcgagtgtta 120
aaagatcgtc cagacatgga ccagactgcc gctttgcaac taccaccagt gggtgagcag 180
attagagagt ttatgggtga gactgtttct aatgcattga ctgccgccaa caccactgaa 240
agcactcaca atatttcaac gagtgatacc ccagcacttc aagctgccga gacgggggca 300
acgtcgaatg caagtgatga gagtatgctt gagaccagga cggttcttaa ccagaatgga 360
attagggaga catccgttga agccttcttt ggcagatctg gattggccac gattatgacc 420
ttggcagcag gtgatgtgaa gacccagtgg accatcaatt tcaatgagtt cgttcagctt 480
agagccaagt tagatttgtt cacttaccta cgatttgaca ttgagttcac tttcgtggcg 540
acatccacga aaaagggtaa gtacaattct gagcccatcc agttacaact gatgtacgtc 600
cccccgggcg ccactcygcc gacggatcaa gacacctatc agtggcagac tgcggcaaac 660
ccctcagtga ttttcat 677
<210> 2
<211> 225
<212> DNA
<213> 人工
<400> 2
qagyvtgwyq tnmvippefp qtaniiclva aqpnfslrvl kdrpdmdqta alqlppvgeq 60
irefmgetvs naltaantte sthnistsdt palqaaetga tsnasdesml etrtvlnqng 120
iretsveaff grsglatimt laagdvktqw tinfnefvql rakldlftyl rfdieftfva 180
tstkkgkyns epiqlqlmyv ppgatxptdq dtyqwqtaan psvif 225
Claims (5)
1.检测G种肠道病毒的免疫荧光试剂,其特征在于:是用荧光染料也可标记的VP1单克隆抗体,所述的VP1单克隆抗体,是利用G种肠道病毒的VP1蛋白,采用杂交瘤单抗制备技术获得;所述的VP1蛋白的氨基酸序列如序列表SEQ ID NO.2所示。
2.根据权利要求1所述的检测G种肠道病毒的免疫荧光试剂,其特征在于:所述的VP1蛋白是由碱基序列如序列表SEQ ID NO.1所示的G种肠道病毒基因VP1表达的。
3.根据权利要求2所述的检测G种肠道病毒的免疫荧光试剂,其特征在于:所述的G种肠道病毒基因VP1为人工合成。
4.根据权利要求1、2或3所述的检测G种肠道病毒的免疫荧光试剂,其特征在于:所述的荧光染料FITC标记。
5.G种肠道病毒直接免疫荧光检测试剂盒,免疫荧光试剂为权利要求1所述的检测G种肠道病毒的免疫荧光试剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610444162.3A CN105974119B (zh) | 2016-06-21 | 2016-06-21 | G种肠道病毒直接免疫荧光试剂及其试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610444162.3A CN105974119B (zh) | 2016-06-21 | 2016-06-21 | G种肠道病毒直接免疫荧光试剂及其试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105974119A true CN105974119A (zh) | 2016-09-28 |
CN105974119B CN105974119B (zh) | 2018-03-09 |
Family
ID=57022022
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610444162.3A Expired - Fee Related CN105974119B (zh) | 2016-06-21 | 2016-06-21 | G种肠道病毒直接免疫荧光试剂及其试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105974119B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110499324A (zh) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | 一种用于鉴定肿瘤新抗原的细菌表达载体及筛选鉴定肿瘤新抗原的方法 |
CN110607404A (zh) * | 2019-10-30 | 2019-12-24 | 广西大学 | 猪肠病毒g型的实时荧光定量pcr检测引物及其试剂盒 |
CN111073859A (zh) * | 2019-12-09 | 2020-04-28 | 东北农业大学 | 一种检测牛细小病毒的双抗体夹心elisa试剂盒及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104945472A (zh) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和包含该器件的检测试剂盒 |
CN105085671A (zh) * | 2015-01-19 | 2015-11-25 | 鄢慧民 | 抗肠道病毒3d蛋白的单克隆免疫球蛋白g抗体及其免疫原性组合物 |
CN105548550A (zh) * | 2016-01-11 | 2016-05-04 | 吉林大学 | 检测e种肠道病毒的免疫荧光试剂及其检测试剂盒 |
-
2016
- 2016-06-21 CN CN201610444162.3A patent/CN105974119B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104945472A (zh) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和包含该器件的检测试剂盒 |
CN105085671A (zh) * | 2015-01-19 | 2015-11-25 | 鄢慧民 | 抗肠道病毒3d蛋白的单克隆免疫球蛋白g抗体及其免疫原性组合物 |
CN105548550A (zh) * | 2016-01-11 | 2016-05-04 | 吉林大学 | 检测e种肠道病毒的免疫荧光试剂及其检测试剂盒 |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110499324A (zh) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | 一种用于鉴定肿瘤新抗原的细菌表达载体及筛选鉴定肿瘤新抗原的方法 |
CN110607404A (zh) * | 2019-10-30 | 2019-12-24 | 广西大学 | 猪肠病毒g型的实时荧光定量pcr检测引物及其试剂盒 |
CN110607404B (zh) * | 2019-10-30 | 2023-02-03 | 广西大学 | 猪肠病毒g型的实时荧光定量pcr检测引物及其试剂盒 |
CN111073859A (zh) * | 2019-12-09 | 2020-04-28 | 东北农业大学 | 一种检测牛细小病毒的双抗体夹心elisa试剂盒及其应用 |
CN111073859B (zh) * | 2019-12-09 | 2021-09-28 | 东北农业大学 | 一种检测牛细小病毒的双抗体夹心elisa试剂盒及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN105974119B (zh) | 2018-03-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104497137B (zh) | 非洲猪瘟病毒毒株通用单克隆抗体及制备方法和应用 | |
NZ582019A (en) | Vaccines containing canine parvovirus genetic variants | |
CN109485703B (zh) | 一种口蹄疫a型结构蛋白vp1抗原表位多肽及其应用 | |
CN103756973B (zh) | 一种草鱼呼肠孤病毒的间接elisa检测试剂盒 | |
CN107973850A (zh) | 重组猪瘟病毒e2蛋白猪源化单克隆抗体及其制备方法和应用 | |
CN110029116B (zh) | 一种分泌表达多抗原表位猪瘟病毒e2基因的重组病毒及制备方法与应用 | |
CN114874995B (zh) | 猪瘟病毒2型Erns蛋白的单克隆抗体杂交瘤细胞株及应用 | |
CN105859842B (zh) | A型口蹄疫病毒单克隆抗体识别的中和表位及其应用 | |
CN112921005A (zh) | 一株杂交瘤细胞株及其产生的犬细小病毒vp2蛋白单克隆抗体和应用 | |
CN105974119A (zh) | G种肠道病毒直接免疫荧光试剂及其试剂盒 | |
CN105548550A (zh) | 检测e种肠道病毒的免疫荧光试剂及其检测试剂盒 | |
CN102406929B (zh) | 一种共表达分子佐剂加强型二价口蹄疫蛋白工程疫苗 | |
US11767356B1 (en) | Canine parvovirus nanobody CPV-VHH-E3 and application thereof | |
CN108872580B (zh) | 一种检测新型鹅细小病毒的胶体金试纸条及其制备方法 | |
CN110257405A (zh) | 牛支原体乙醇脱氢酶基因及其编码蛋白与应用 | |
CN113512098B (zh) | 鉴别猪瘟病毒和牛病毒性腹泻病毒血清抗体间接elisa方法及其应用 | |
CN109374886A (zh) | 牛传染性鼻气管炎病毒抗体检测试剂盒及其应用 | |
CN104744595B (zh) | 抗草鱼出血病病毒工程蛋白tat‑vp7‑tat及制备方法和应用 | |
CN102370976B (zh) | 猪流感病毒和猪口蹄疫病毒的混合病毒样颗粒、制备方法和应用 | |
CN105968196A (zh) | G种肠道病毒检测抗体双夹心elisa检测试剂盒 | |
CN106397602A (zh) | 一种分子佐剂加强型鸡马立克氏病蛋白工程疫苗 | |
CN107271667B (zh) | 蓝舌病病毒型特异性竞争elisa检测试剂盒及其应用 | |
CN101979406B (zh) | 南非ⅱ型口蹄疫多抗原表位蛋白、其制备方法及应用 | |
US8609404B2 (en) | Vaccines containing canine parvovirus genetic variants | |
CN107602674A (zh) | 可溶性i型鸭肝炎病毒3c蛋白的制备方法及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | 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: 20180309 |