CN116284431A - Preparation and application of novel coronavirus polyclonal antibody - Google Patents
Preparation and application of novel coronavirus polyclonal antibody Download PDFInfo
- Publication number
- CN116284431A CN116284431A CN202210915208.0A CN202210915208A CN116284431A CN 116284431 A CN116284431 A CN 116284431A CN 202210915208 A CN202210915208 A CN 202210915208A CN 116284431 A CN116284431 A CN 116284431A
- Authority
- CN
- China
- Prior art keywords
- aac
- ggt
- ccg
- antibody
- novel coronavirus
- 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
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 135
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 141
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 136
- 238000001514 detection method Methods 0.000 claims abstract description 125
- 238000000034 method Methods 0.000 claims abstract description 67
- 239000000427 antigen Substances 0.000 claims abstract description 57
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Substances C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 102000036639 antigens Human genes 0.000 claims abstract description 55
- 108091007433 antigens Proteins 0.000 claims abstract description 55
- 238000002965 ELISA Methods 0.000 claims abstract description 53
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 45
- 238000000746 purification Methods 0.000 claims abstract description 21
- 238000003118 sandwich ELISA Methods 0.000 claims abstract description 21
- 238000010828 elution Methods 0.000 claims abstract description 15
- 239000002671 adjuvant Substances 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 9
- 230000002163 immunogen Effects 0.000 claims abstract description 7
- 238000011587 new zealand white rabbit Methods 0.000 claims abstract description 7
- 244000052769 pathogen Species 0.000 claims abstract description 5
- 210000002966 serum Anatomy 0.000 claims description 47
- 230000003053 immunization Effects 0.000 claims description 43
- 238000002649 immunization Methods 0.000 claims description 43
- 239000013612 plasmid Substances 0.000 claims description 40
- 238000010790 dilution Methods 0.000 claims description 39
- 239000012895 dilution Substances 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 33
- 241001678559 COVID-19 virus Species 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 30
- 238000001962 electrophoresis Methods 0.000 claims description 27
- 230000000903 blocking effect Effects 0.000 claims description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 21
- 239000004202 carbamide Substances 0.000 claims description 21
- 238000000502 dialysis Methods 0.000 claims description 21
- 241000894006 Bacteria Species 0.000 claims description 20
- 230000014509 gene expression Effects 0.000 claims description 19
- 230000001965 increasing effect Effects 0.000 claims description 19
- 238000010257 thawing Methods 0.000 claims description 18
- 239000012634 fragment Substances 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 229940088598 enzyme Drugs 0.000 claims description 12
- 210000003000 inclusion body Anatomy 0.000 claims description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 11
- 238000007710 freezing Methods 0.000 claims description 11
- 230000008014 freezing Effects 0.000 claims description 11
- 238000003860 storage Methods 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 9
- 240000000220 Panda oleosa Species 0.000 claims description 8
- 235000016496 Panda oleosa Nutrition 0.000 claims description 8
- 238000002835 absorbance Methods 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 102000005962 receptors Human genes 0.000 claims description 8
- 108020003175 receptors Proteins 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 claims description 7
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims description 7
- 229940096437 Protein S Drugs 0.000 claims description 7
- 102000029301 Protein S Human genes 0.000 claims description 7
- 101710198474 Spike protein Proteins 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 claims description 7
- 108010052968 leupeptin Proteins 0.000 claims description 7
- 239000006166 lysate Substances 0.000 claims description 7
- 230000028327 secretion Effects 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000001042 affinity chromatography Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000001976 enzyme digestion Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 229920003023 plastic Polymers 0.000 claims description 5
- 239000004033 plastic Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 238000001262 western blot Methods 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 235000020183 skimmed milk Nutrition 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 108700002856 Coronavirus Envelope Proteins Proteins 0.000 claims description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 239000004568 cement Substances 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 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 claims description 2
- 229930027917 kanamycin Natural products 0.000 claims description 2
- 229960000318 kanamycin Drugs 0.000 claims description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 2
- 229930182823 kanamycin A Natural products 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000012300 Sequence Analysis Methods 0.000 claims 2
- 241001052560 Thallis Species 0.000 claims 1
- 210000004201 immune sera Anatomy 0.000 claims 1
- 229940042743 immune sera Drugs 0.000 claims 1
- 229950000964 pepstatin Drugs 0.000 claims 1
- 108010091212 pepstatin Proteins 0.000 claims 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 claims 1
- 210000002345 respiratory system Anatomy 0.000 claims 1
- 239000003053 toxin Substances 0.000 claims 1
- 231100000765 toxin Toxicity 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 17
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 229910001453 nickel ion Inorganic materials 0.000 abstract description 3
- 230000000241 respiratory effect Effects 0.000 abstract description 3
- 230000009920 chelation Effects 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 25
- 239000000523 sample Substances 0.000 description 14
- 208000015181 infectious disease Diseases 0.000 description 10
- 238000004153 renaturation Methods 0.000 description 10
- 241000700605 Viruses Species 0.000 description 9
- 238000011161 development Methods 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 101710172711 Structural protein Proteins 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 208000025721 COVID-19 Diseases 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 101800001632 Envelope protein E Proteins 0.000 description 4
- 244000309467 Human Coronavirus Species 0.000 description 4
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 241000598171 Human adenovirus sp. Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 3
- 101710141454 Nucleoprotein Proteins 0.000 description 3
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 3
- 241000725643 Respiratory syncytial virus Species 0.000 description 3
- 241000710799 Rubella virus Species 0.000 description 3
- 241000315672 SARS coronavirus Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000012501 chromatography medium Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 2
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 2
- 101710139375 Corneodesmosin Proteins 0.000 description 2
- 102100031673 Corneodesmosin Human genes 0.000 description 2
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 2
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 208000037847 SARS-CoV-2-infection Diseases 0.000 description 2
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- -1 pepstatin Chemical compound 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 241000008904 Betacoronavirus Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 241000662429 Fenerbahce Species 0.000 description 1
- 241000711467 Human coronavirus 229E Species 0.000 description 1
- 241001109669 Human coronavirus HKU1 Species 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 241001428935 Human coronavirus OC43 Species 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 101000667982 Severe acute respiratory syndrome coronavirus 2 Envelope small membrane protein Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 108010059277 canatoxin Proteins 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 230000036387 respiratory rate Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014599 transmission of virus Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- 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
- 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
- C12N2770/20051—Methods of production or purification of viral material
-
- 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
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses preparation and application of a novel coronavirus polyclonal antibody, and relates to the fields of genetic engineering technology, polyclonal antibody preparation and purification technology and diagnostic reagents. Expressing the novel coronavirus multi-antigen epitope chimeric protein by using escherichia coli, and purifying the novel coronavirus multi-antigen epitope chimeric protein by adopting a nickel ion chelation and chromatography-imidazole gradient elution method. The chimeric Protein is used as an immunogen to be matched with Freund's adjuvant to immunize New Zealand white rabbits to prepare rabbit-derived polyclonal antibodies, the titer and the specificity of the polyclonal antibodies are evaluated, and a Protein A column is used for purifying novel coronavirus polyclonal antibodies. The prepared novel coronavirus polyclonal antibody and HRP marked polyclonal antibody are respectively used as a capture antibody and a detection antibody, and a double-antibody sandwich ELISA detection reagent is established. The minimum detection limit of the ELISA detection reagent is 0.039 ng/. Mu.L, the linear range is 0.039-1.25 ng/. Mu.L, and the detection of other common respiratory pathogens is negative, so that the ELISA detection reagent has better specificity. ELISA detection reagents are established for detection of novel coronavirus antigens.
Description
Technical Field
The invention discloses preparation and application of a novel coronavirus polyclonal antibody, and relates to the fields of genetic engineering technology, polyclonal antibody preparation and purification technology and diagnostic reagents. The invention uses two connecting proteins to connect the extracellular region segment (16 amino acids in length) of envelope protein E of novel coronavirus (SARS-CoV-2), N-terminal segment (177 amino acids in length) of nucleocapsid protein N and receptor binding domain RBD segment (223 amino acids in length) of spike protein S in series, so as to construct new coronavirus antigen epitope chimeric protein sequence. Preparing chimeric proteins containing a plurality of novel coronavirus antigen epitopes by using a genetic engineering technology, preparing polyclonal antibodies by using the chimeric proteins as immunogens to be matched with Freund's adjuvant to immunize New Zealand white rabbits, evaluating the titer and the specificity of the polyclonal antibodies, and purifying the polyclonal antibodies by using a Protein A column. The purified polyclonal antibody is used for preparing HRP marked polyclonal antibody, and the polyclonal antibody and the HRP marked polyclonal antibody are respectively used as capture antibody and detection antibody, so as to establish a double-antibody sandwich ELISA detection method which can be used for detecting SARS-CoV-2 antigen.
Background
The current and popular new type of coronavirus pneumonia (covd-19) has changed the world in an unprecedented way, and brings great threat to the life and property safety of people and great challenge to the social and economic development. The main sources of infection for the novel coronaviruses are diagnosed patients and asymptomatic infected persons, which are transmitted from person to person mainly by respiratory droplets and aerosols, however, studies have also shown that SARS-CoV-2 can be transmitted by contaminated objects. SARS-CoV-2 incubation period is 1-14 days, most 3-7 days, and there is infectivity in incubation period. About 81% of patients with COVID-19 have mild symptoms after SARS-CoV-2 infection; about 14% of patients have severe symptoms such as dyspnea, high respiratory rate, low in vivo blood oxygen saturation; about 5% of patients, particularly those over 60 years old or with other diseases, often progress to criticality; about 3.4% of patients die from respiratory failure or multiple organ failure. Therefore, early, rapid and accurate identification of patients infected with SARS-CoV-2 and timely treatment of patients are important to prevent continuous transmission of viruses.
SARS-CoV-2 is assigned to a new species of the genus beta coronavirus, which has a homology of 79.6% with SARS coronavirus. SARS-CoV-2 genome is 29.8-29.9 kb linear non-segmented single-stranded positive strand RNA, comprising 14 open reading frames, with the typical morphological and genomic characteristics of coronaviruses. SARS-CoV-2 is composed mainly of 4 structural proteins, which play an important role in viral assembly and infection of the host: spike protein S is responsible for binding to host cell receptors, membrane protein M maintains the basic morphology of the virion, envelope protein E is involved in viral assembly, release and pathogenesis, nucleocapsid protein N binds viral genomic RNA in different forms, wherein S protein and N protein are their major antigenic proteins. SARS-CoV-2 is the seventh human coronavirus (HCoV) known to be responsible for human disease, and the other six types of HCoVs include HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, where SARS-CoV-2, SARS-CoV, and MERS-CoV are considered highly pathogenic. In fact, although the mortality rate of COVID-19 is lower than that of SARS and MERS, the number of infections and deaths of COVID-19 is much higher than that of SARS and MERS.
Currently, there are four main methods for detecting SARS-CoV-2 infection: (1) Isolating a virus, such as a Vero cell culture, by inoculating a biological sample of a patient into the cell culture, which requires biosafety class 3 laboratory facilities; (2) Molecular biology techniques such as reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and high throughput sequencing, are powerful tools for the discovery of pathogens; (3) Serological detection, antigen and antibody detection by enzyme-linked immunosorbent assay (ELISA), and immunofiltration, immunochromatography and chemiluminescent immunoassay; (4) Antigen detection with antibodies specific for SARS-CoV-2 antigen.
In general, the human immune system is able to elicit humoral and cellular immune responses and gradually control infection and eliminate viruses when infected by the virus. When viremia occurs in early infection, virus nucleic acid can be detected from serum and nasal and pharyngeal secretions of patients, so that the nucleic acid detection is very suitable for early infection, but the nucleic acid detection method is complex, long in time consumption and high in infection risk of detection personnel, and is not suitable for rapid detection of a basic layer and a site. In addition, specific IgM starts to be produced in patients about 7 days after the infection of the novel coronavirus, and specific IgG starts to be produced 14 days after the infection, so that the antibody detection reagent is not suitable for detection of the early stage of the infection of the novel coronavirus, and a high false negative rate exists. In the new coronavirus antigen detection reagent products, most of the detection reagent products are a colloidal gold method and a latex method, N protein of a single new coronavirus is mainly used as a target antigen, the sensitivity is 75-98%, and certain false negative conditions exist. Thus, the use of a novel coronaantibody recognizing a plurality of structural protein epitopes of a novel coronavirus is expected to increase the sensitivity of detection of the novel coronavirus.
Disclosure of Invention
The invention aims to develop a novel coronavirus polyclonal antibody which is used for detecting antigens of novel coronaviruses. Of the four structural proteins of the novel coronavirus (SARS-CoV-2), spike protein S and nucleocapsid protein N have multiple epitopes, wherein the receptor binding domain fragment (RBD) on the S protein more directly mediates the binding of viral particles to the host cell receptor angiotensin converting enzyme-2 (ACE-2), which induces the body to produce multiple specific antibodies; in addition, previous studies on human coronaviruses have found that deletion of the envelope protein E results in attenuation of the virus, indicating that the E protein is also a potential target for antiviral and vaccine design. Therefore, these three structural proteins are ideal proteins for developing novel crown polyclonal antibodies and antigen detection reagents.
After analysis by software, a novel coronavirus epitope chimeric protein sequence was designed, comprising an extracellular region fragment (16 amino acids in length) of SARS-CoV-2 envelope protein E, an N-terminal fragment (177 amino acids in length) of nucleocapsid protein N, and a receptor binding domain RBD fragment (223 amino acids in length) of spike protein S. The chimeric protein sequence has 422 amino acids in total length and 46.52kD molecular weight, and is expressed in the form of inclusion bodies through an escherichia coli prokaryotic expression system by using pET-28a (+) as a carrier plasmid, and the novel coronavirus multi-antigen epitope chimeric protein is obtained after purification and gradient dialysis renaturation through denaturation, nickel ion chelation and chromatography-imidazole gradient elution methods. The chimeric Protein is used as an immunogen to be matched with Freund's adjuvant to immunize New Zealand white rabbits to prepare rabbit-derived polyclonal antibodies, the polyclonal antibodies capable of recognizing a plurality of structural Protein epitopes of the new coronavirus are obtained through Protein A column purification, the prepared polyclonal antibody is used for developing a double-antibody sandwich ELISA detection method, the lowest detection limit and the specificity of the method are evaluated after the dilution combination of the optimal capture antibody and the detection antibody, the optimal blocking liquid type and the optimal blocking time are determined, and the influence of the storage condition of a sample on the lowest detection limit of the method is compared.
The preparation and application of the novel coronavirus (SARS-CoV-2) polyclonal antibody are carried out by adopting the following steps:
the preparation and application of a novel coronavirus polyclonal antibody relates to a patent related to earlier application: a novel coronavirus epitope chimeric protein and its preparation and application (patent application number: 2021110369027) are disclosed, wherein the chimeric protein is formed by connecting an extracellular region segment of a novel coronavirus envelope protein E, an N-terminal segment of a nucleocapsid protein N and a receptor binding domain RBD segment of spike protein S, the protein segments are connected by two glycine and one serine, and the total length of the chimeric protein is 422 amino acids.
Sequence of novel coronavirus epitope chimeric protein:
according to the previously filed patent: the novel coronavirus epitope chimeric protein and the preparation and application thereof (patent application number: 2021110369027) are characterized in that a novel coronavirus epitope chimeric protein gene sequence designed in the preparation and application thereof (patent application number: 2021110369027) is introduced into a vector plasmid pET-28a (+) after chemical synthesis, and the amino acid sequence and the gene sequence of the novel coronavirus epitope chimeric protein are shown as follows:
amino acid sequence of novel coronavirus epitope chimeric protein:
The encoding gene of the novel coronavirus antigen epitope chimeric protein has the nucleotide sequence as follows: DNA sequence of chemically synthesized novel crown chimeric protein (1281 bp)
(EcoRI)
(SalI)
The preparation and application of the novel coronavirus polyclonal antibody use escherichia coli to express novel coronavirus antigen epitope chimeric protein, and the specific preparation method is as follows:
1. construction of chimeric protein recombinant plasmid expressing SARS-CoV-2 epitope:
plasmid pET-28a (+) (product of Novagen, U.S.A.), was extracted, digested with EcoRI and SalI, and after electrophoresis, the digested plasmid fragment was recovered and dissolved in deionized water. SARS-CoV-2 antigen epitope chimeric protein gene fragment chemically synthesized by EcoRI and SalI double enzyme digestion, and after electrophoresis recovery, dissolved in deionized water. The DNA fragment after the enzyme digestion is inserted between EcoRI and SalI sites in a vector pET-28a (+) to express chimeric proteins containing novel coronavirus epitopes.
2. Screening and identification of recombinant plasmids (double restriction identification of recombinant plasmids):
the recombinant plasmid was transformed into E.coli BL21 (DE 3), and LB plates containing kanamycin (50. Mu.g/mL) were plated out and left at 37℃overnight. The transformed colonies were randomly picked the next day to extract plasmids, and double digestion with EcoRI and HindIII was used to verify that 1.0% agarose gel electrophoresis showed that the excised target gene fragment was consistent with the expected size. Meanwhile, the plasmid containing the gene fragment is subjected to DNA sequencing analysis, and the sequence is completely correct:
3. Screening and identifying engineering bacteria expressing fusion proteins:
inoculating positive transformant containing recombinant plasmid into test tube containing 4mLLB culture medium containing Canatoxin 50 mug/mL, shake culturing overnight at 37 deg.C, adding IPTG to final concentration of 0.2mmol/L the next day, shake culturing and inducing for 4 hr, centrifuging to collect thallus, performing SDS-PAGE detection, expressing SARS-CoV-2 chimeric protein with relative molecular weight of 46.52kD, and selecting strain with highest expression amount for subsequent test.
4. Purification of SARS-CoV-2 epitope chimeric protein:
(1) Ultrasonic cleavage of SARS-CoV-2 epitope chimeric protein engineering bacteria:
the high expression strain is subjected to induced expression after inoculation, expansion and strain storage, engineering bacteria for induced expression of chimeric proteins are subjected to centrifugation and bacteria collection, the centrifugation condition is 8000rpm, 15min and 4 ℃, 1/10 volume of bacterial lysate of an original culture solution is prepared, the lysate contains 20mmol/L PB and 5% glycerol, the pH value is 7.4, lysozyme and Leupeptin, pepstatin, pmsf are added and uniformly mixed, and the centrifuged bacteria are subjected to weight suspension by using the bacterial lysate. The ice bath ultrasonic bacteria breaking, collecting supernatant and sediment after centrifugation, respectively reserving samples, and detecting the expression form of the chimeric protein by 12% SDS-PAGE electrophoresis, wherein the electrophoresis result shows that the chimeric protein is mainly expressed in the form of inclusion bodies.
(2) Washing and denaturation of inclusion bodies:
the inclusion bodies were washed with 200mL of 1mol/L urea, the urea solution was prepared with 1 XPBS and 1% Triton X-100 was added, the precipitate remained after washing and centrifugation at 8000rpm at 4℃for 15min, the precipitate was denatured with 200mL of 8mol/L urea, the 8mol/L urea solution was prepared with 1 XPBS and the supernatant was collected after centrifugation at 8000rpm at 4℃for 15 min.
(3) Chimeric proteins bind to cement:
to a 12mL plastic column, 5mL of High-Affinity Ni-NTAResin was added, and the gel was washed 5 times with 1 XPBS to remove residual ethanol from the gel. The 8mol/L urea denatured supernatant was filtered with 0.45 μm filter paper and mixed with the prepared gum, and stirred and combined for 1-2h under ice bath conditions.
(4) Elution of chimeric proteins:
protein and gum mixtureCollecting flow through peaks by using a plastic chromatographic column, fully washing the column by using 8mol/L urea as a balance liquid, sequentially using 20, 50, 100, 200 and 500mmol/L imidazole eluate, preparing the imidazole eluate by using 8mol/L urea, eluting for 15min each time, collecting proteins of each eluting peak, and detecting OD of the proteins 280 Value up to protein OD 280 Below 0.1, the imidazole eluent is changed into an imidazole eluent with higher concentration. Each elution peak protein was detected by 12% SDS-PAGE electrophoresis to determine which elution peak contained the expressed SARS-CoV-2 epitope chimeric protein. Electrophoresis results show that the purity and the concentration of the novel coronavirus epitope chimeric protein in the imidazole elution protein peaks of 20, 50 and 100mmol/L are higher.
(5) Dialysis renaturation of chimeric proteins:
taking a dialysis bag with proper size, placing the dialysis bag into deionized water, heating for 5min by microwaves, taking out, cooling, pouring the eluted denatured protein into the dialysis bag, clamping two ends of the dialysis bag, and sequentially carrying out dialysis renaturation by using 6, 4, 3, 2, 1mol/L urea solution and 1 XPBS (glycerol with the final concentration of 10%, EDTA with the final concentration of 10mM/L and DTT with the concentration of 1mM/L are added into each gradient), wherein the volume of the dialysate used for each dialysis is 2L, and the dialysis time is 3h. The novel coronavirus epitope chimeric protein with higher purity and concentration is obtained after dialysis renaturation.
(6) Determination of chimeric protein concentration:
the protein concentration was measured by BCA method, a standard curve was drawn, absorbance values of the samples were brought into the curve, and the concentration of the novel crown chimeric protein was calculated to be 0.673mg/ml.
The preparation and application of the novel coronavirus polyclonal antibody uses the novel coronavirus epitope chimeric protein as an immunogen, the novel coronavirus epitope chimeric protein is matched with Freund's adjuvant to immunize New Zealand white rabbits, rabbit serum is taken for purification, and the corresponding rabbit-derived polyclonal antibody is prepared, and the polyclonal antibody is characterized in that the novel coronavirus epitope chimeric protein can be identified, and the preparation and purification methods of the polyclonal antibody are as follows:
1. Preparation of novel coronavirus polyclonal antibodies:
immunization with novel coronavirus chimeric proteinsEach 500 mug of the antigen is matched with Freund's adjuvant to immunize rabbits, the whole immunization is carried out according to a pre-designed scheme, and the specificity of antiserum is detected through Western-Blot. Blood is taken after each immunization to detect the antibody OD 450 The value was analyzed for secretion of serum antibodies with increasing number of immunizations. Serum after the fourth immunization is subjected to gradient dilution, the titer of the antiserum is detected by an ELISA method, and the detection result shows that the OD of the serum antibody is increased along with the increase of the immunization times 450 The value was also increasing, and the serum antibody titer reached the highest after the fourth immunization, 1:1280000.
2. purification of novel coronavirus polyclonal antibodies:
separating and purifying polyclonal antibody from serum after four times of immunization by Protein A column affinity chromatography, standing blood at room temperature for 1h after each blood sampling, standing at 4deg.C for 4h for layering, centrifuging at 12000rpm for 15min, and sucking upper serum layer in a sterile console for use. Serum was dialyzed against 1 XPBS and diluted overnight to bind to the medium, and the antibody was eluted by changing the pH of the solution to alter the affinity of the medium for the antibody. The 4 times of serum purified by the Protein A column is respectively sampled for gel electrophoresis analysis, and the electrophoresis result shows that the concentration of the polyclonal antibody is continuously increased along with the increase of the immunization times, and the concentration of the polyclonal antibody is 1.34mg/mL detected by the BCA method.
The novel coronavirus rabbit-derived polyclonal antibody prepared by the invention establishes a double-antibody sandwich ELISA detection method, and evaluates the minimum detection limit and the specificity of the ELISA method, and specifically comprises the following steps:
(1) Dialyzing the purified rabbit polyclonal antibody with 1 XPBS, detecting pH value of the polyclonal antibody after dialysis to reach a proper range of 6.5-8.5, diluting the polyclonal antibody to 1 μg/μL with 1 XPBS, subpackaging, and standing at-20deg.C for use.
(2) And preparing the HRP-labeled polyclonal antibody from the dialyzed polyclonal antibody solution by using an HRP-labeled antibody kit.
(3) The purified rabbit polyclonal antibody is used as a capture antibody, the HRP-labeled rabbit polyclonal antibody is used as a detection antibody, and the optimal dilution combination of the capture antibody and the detection antibody is obtained by a square method, wherein the dilution combination is as follows: capture antibody dilution 1:6400, detection antibody dilution 1:800.
(4) Three sealing liquids are arranged: 3% BSA, 5% BSA and 5% skim milk powder were tested using this ELISA method and the optimal blocking solution type was determined to be 3% BSA based on the P/N value.
(5) Setting 6 closing times: 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, using this ELISA method to detect, the optimal blocking time was determined to be 2h based on the P/N value.
(6) The initial concentration of novel coronavirus epitope chimeric protein ENSRBD is 0.5mg/mL according to 1: and (3) carrying out gradient dilution on the ELISA (enzyme-linked immunosorbent assay) according to the ratio of 25-1:51200, wherein the lowest detection limit of the ELISA is 0.039 ng/. Mu.L according to the P/N value of >2.1, and the linear range of the ELISA is 0.039-1.25 ng/. Mu.L by taking the logarithm of the absorbance value and the chimeric protein concentration value respectively.
(7) The established ELISA detection method is used for respectively detecting novel coronavirus ENSRBD proteins and other common respiratory pathogen antigens, and the detection results are judged according to the P/N values so as to evaluate the specificity of the established ELISA detection method, and the detection results show that the established ELISA detection method has better specificity. The specificity calculation method here is as follows: specificity = number of antigen types that the method detects as negative/number of antigen types that are actually negative.
(8) Effect of sample storage conditions on method detection limits:
three room temperature times were set: 25 ℃ for 1h, 4h and 8h; three freeze thawing times are set: repeatedly freezing and thawing for 1 time, 3 times and 5 times. The established ELISA method is used for detecting the new crown chimeric protein diluted in a gradient way, the influence of the storage condition of the sample on the detection method is analyzed, and the detection result shows that the detection result is improved along with the extension of the room temperature standing time and the increase of the repeated freezing and thawing times, so that the ELISA method is used for avoiding the long-time room temperature standing and repeated freezing and thawing of the sample.
The polyclonal antibody corresponding to the novel coronavirus epitope chimeric protein is used for detecting SARS-CoV-2 antibody or antigen.
The novel coronavirus double-antibody sandwich ELISA detection method is applied to the field of novel coronavirus antigen detection.
The prepared polyclonal antibody corresponding to the novel coronavirus epitope chimeric protein is used for detecting SARS-CoV-2 antibody or antigen, etc.
The established double-antibody sandwich ELISA method is used in the field of novel coronavirus antigen detection.
English abbreviations description: SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2): novel coronaviruses; EDTA: tetramethyl ethylenediamine; leupeptin: leupeptin; pepstatin: gastric inhibitory peptidase solution; pmsf: benzyl sulfonyl fluoride; IPTG: isopropyl thiogalactoside; SDS: sodium dodecyl sulfonate; PAGE: polyacrylamide gel electrophoresis; PB: phosphate buffer; kD: kilodaltons.
The invention has the advantages compared with the prior art
The novel coronavirus polyclonal antibody and the double-antibody sandwich ELISA method prepared by the invention have more advantages:
1. the detection of the novel coronavirus is mainly nucleic acid detection, antigen detection is supplemented, most of the existing novel coronavirus antigen detection reagent products are a colloidal gold method and a latex method, N protein of a single novel coronavirus is mainly used as a target antigen, the detection sensitivity is 75% -98%, certain false negative conditions exist, and the use of novel coronavirus antibodies capable of recognizing a plurality of novel coronavirus structural protein epitopes is expected to improve the detection sensitivity of the novel coronavirus antigens.
2. The polyclonal antibody used in the double antibody sandwich ELISA method is a patent applied according to the previous subject group: a novel coronavirus antigen epitope chimeric protein and preparation and application thereof (patent application number: 20211110369027) are provided, wherein the novel coronavirus antigen epitope chimeric protein is prepared by taking the novel coronavirus antigen epitope chimeric protein as an immunogen and immunizing rabbits with Freund's adjuvant, and the chimeric protein ENSRBD is expressed by using a prokaryotic expression system, and the prokaryotic expression system has the advantages of short production period, low cost, clear genetic background, strong anti-pollution capability, high expression quantity of exogenous genes and the like. The chimeric protein mainly exists in the form of inclusion bodies, the expression quantity is high, the yield of the soluble protein after renaturation treatment is high, and the subsequent dilution is convenient for carrying out animal immunity experiments and the preparation work of corresponding monoclonal antibodies and polyclonal antibodies.
3. Compared with a monoclonal antibody, the polyclonal antibody has the advantages of short preparation period, low cost, direct purification from animal serum, and capability of recognizing more antigen epitopes, thereby being beneficial to improving the sensitivity of an antigen detection method.
Drawings
The invention will be further described with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of the construction of a recombinant plasmid expressing a novel coronavirus epitope chimeric protein, and a chemically synthesized gene fragment of interest is inserted between EcoRI and SalI sites in the vector pET-28a (+).
FIG. 2 shows the results of double digestion identification of recombinant plasmids expressing novel coronavirus epitope chimeric proteins, lane 2 shows the recombinant plasmids after double digestion, and specific bands appear at 1281bp, consistent with the expected size, indicating that the recombinant plasmids are constructed correctly.
FIG. 3 is an SDS-PAGE electrophoresis of the constructed recombinant plasmid transformed into E.coli, and a high-yield strain selected. The six recombinant bacteria all express chimeric proteins with the relative molecular weight of 46.52kDa, namely the positions marked by arrows in the figure, and the No. 4 high-expression strain is selected for subsequent experiments.
FIG. 4 shows the result of SDS-PAGE analysis of novel coronavirus epitope chimeric protein expression, lane 2 is the disrupted supernatant, and lane 3 is the disrupted pellet, indicating that the chimeric protein is expressed mainly in the form of inclusion bodies.
FIG. 5 is a SDS-PAGE analysis of imidazole gradient elution of novel coronavirus epitope chimeric proteins eluted predominantly in 20, 50 and 100mmol/L imidazole eluate.
FIG. 6 shows the SDS-PAGE analysis result after renaturation of novel coronavirus epitope chimeric proteins, and lanes 2-3 show renatured chimeric proteins.
FIG. 7 is a standard graph of BCA kit for detecting novel coronavirus epitope chimeric protein concentration.
FIG. 8 shows the result of detection of the specificity of antisera after immunization of the novel coronavirus epitope chimeric protein, and lane 1 shows the novel coronavirus epitope chimeric protein.
FIG. 9 is a graph showing ELISA results of antiserum secretion after each immunization of novel coronavirus epitope chimeric proteins, serum antibody OD with increasing number of immunization 450 The value is also increasing.
FIG. 10 shows ELISA results of rabbit antiserum titers after four-immunization of novel coronavirus epitope chimeric proteins, and the antibody titers after four-immunization reach 1:1280000.
FIG. 11 shows the results of electrophoresis of the rabbit polyclonal antibody purified by Protein A column, lanes 1-4 are the purified polyclonal antibodies after 1-4 immunizations, respectively, with the concentration of polyclonal antibodies increasing continuously with increasing number of immunizations.
FIG. 12 is a standard graph of the BCA kit for detecting rabbit polyclonal antibody concentration.
FIG. 13 is a graph showing the results of a linear range assay for a double antibody sandwich ELISA method with a linear range of 0.039-1.25 ng/. Mu.L.
FIG. 14 shows the result of specificity determination of the double antibody sandwich ELISA detection method, and the result shows that the established ELISA detection method has better specificity.
Table 1 is the ELISA results of screening the best capture antibody and detection antibody dilution combinations, best combinations being: the capture antibody dilution was 1:6400 and the detection antibody dilution was 1:800.
Table 2 shows ELISA results for screening the optimal blocking solution type, which was 3% BSA.
Table 3 shows ELISA results for screening for optimal blocking time, which was 2h.
Table 4 shows the determination results of the minimum detection limit of the double antibody sandwich ELISA, and the minimum detection limit of the method is 0.039 ng/. Mu.L.
Table 5 shows the effect of the room temperature standing time on the minimum detection limit of the method, and shows a tendency to increase with the increase of the minimum detection limit of the room temperature standing time.
Table 6 shows the effect of the number of repeated freeze thawing on the minimum detection limit of the method, and shows a trend of increasing with the increase of the minimum detection limit of the number of repeated freeze thawing.
Detailed Description
Detailed description of embodiments of the invention:
preparation and purification of novel coronavirus epitope chimeric protein
According to the previously filed patent: a novel coronavirus antigen epitope chimeric protein and its preparation and application (patent application number: 2021110369027) are disclosed, which comprise N-terminal extracellular region segment of envelope protein E, N-terminal segment of nucleocapsid protein N and receptor binding domain RBD segment of spike protein S. The full length of the chimeric protein is 422 amino acids, the full length gene sequence of the chimeric protein is chemically synthesized, and cloned to EcoRI and SalI sites in plasmid pET-28a (+) to express the novel coronavirus epitope chimeric protein. The recombinant plasmid is transformed into escherichia coli BL21 (DE 3), engineering bacteria which efficiently express novel coronavirus epitope chimeric proteins are obtained through screening, the expressed novel coronavirus chimeric proteins account for more than 60% of the total bacterial protein, the chimeric proteins are purified by adopting a nickel ion chelating and chromatography-imidazole gradient elution method, and the novel soluble coronavirus epitope chimeric proteins are obtained through gradient dialysis renaturation.
Materials and methods
1. Bacterial species and plasmid: host bacterium BL21 (DE 3) and expression vector pET-28a (+) are products of Novagen corporation in America.
2. Molecular biological agents: restriction endonucleases EcoRI and SalI, T4 DNA ligase and IPTG are products of Takara company; isopropyl thio- β -D-galactoside (IPTG) is a Merck company product; dithiothreitol (DTT) is a product of BIOSHARP corporation; leupeptin (leupeptin), PMSF, imidazole, glycerol are products of the biological company; the gastric inhibitory peptidase solution is a source leaf biological product; kanamycin sulfate is a product of Adamas company, and a protein marker is a product of Takara company; the plasmid extraction kit and the kit for recovering DNA fragments from agarose gel are products of Takara company; the kit for measuring the protein concentration by the BCA method is a Biyun Tian company product; express Cast PAGE the color gel rapid kit is a product of New Saimei biosciences, inc.; the High Affinity Ni-NTA Affinity chromatography medium is a product of Kirschner company; other reagents are homemade or imported analytically pure reagents.
3. Synthesis of gene fragments: the recombinant plasmid was synthesized by Nanjing Jinsri company, and the construction diagram of the recombinant plasmid is shown in FIG. 1.
DNA sequence verification: sequencing verification was performed by the company nanjing gold sry.
5. Double enzyme digestion identification of recombinant plasmid:
the E.coli BL21 after transformation is coated on LB culture medium plate containing kana resistance, single bacteria are selected for culturing, bacteria are collected and plasmids are extracted, the extracted recombinant plasmids are subjected to double digestion by EcoR I and HindIII, and the digestion products are subjected to agarose gel electrophoresis of 1 percent, as shown in figure 2.
6. E.coli is transformed by the recombinant plasmid, and high expression strains are selected:
each plate of Kana-resistant solid LB selection dishes was coated with 50. Mu.l, 100. Mu.l, 150. Mu.l, 200. Mu.l of the transformed bacterial solutions, and incubated at 37℃for 12-16h until apparent plaques appeared. The following day, 6 single colonies were selected from the dishes and inoculated into tubes containing 4ml LB liquid medium (Kana 4. Mu.l added), and E.coli BL21 (DE 3) containing pET-28a (+) empty plasmid was further inoculated into tubes containing Kana-resistant 4ml LB liquid medium (2 tubes, used as negative control), and cultured overnight at 37℃at 200 rpm. The next day, 8 tubes were subjected to an expansion culture (Kana resistance was previously added) at 37℃for 1 hour at 200 rpm. After the strain is stored and marked, adding IPTG 1 μl into the residual strain liquid, and carrying out induction expression for 4 hours at 25 ℃ and 200rpm, sampling each tube for electrophoresis, and selecting the high-expression strain according to the electrophoresis result for subsequent experiments, wherein the electrophoresis result is shown in figure 3.
7. Induced expression and expression form analysis of novel coronavirus epitope chimeric protein engineering bacteria:
the high expression strain was inoculated into two flasks containing 200mL of LB fresh liquid medium, which was pre-supplemented with Kana resistance to 50. Mu.g/mL and incubated overnight in a shaker at 37 ℃. The next day, the bacterial liquid is expanded into 8 triangular flasks containing 200mL of LB liquid medium, kana resistance is added to 50 mug/mL in advance, the culture medium is placed in a shaking table at 37 ℃ for shaking culture for 1.5 hours, IPTG with the final concentration of 0.2mmol/L is added after the bacterial liquid is reserved, and induced expression is carried out for 4 hours at 25 ℃. 1600mL engineering bacteria inducing to express chimeric proteins are centrifugated at 8000rpm and 15min and 4 ℃ for bacterial recovery, bacterial cells are redissolved by 200mL bacterial lysate, the lysate contains 20mmol/LPB and 5% glycerol, the pH is 7.4 (after lysozyme and Leupeptin, pepstatin, pmsf are added and evenly mixed), the bacteria are ultrasonically broken by ice bath for 75 times, each time is broken for 4s, the interval time is 4s, and then the supernatant and the precipitate are collected by centrifugation at 8000rpm and 4 ℃ for 15 min. The supernatant and the pellet were separately sampled and subjected to 12% SDS-PAGE electrophoresis, and the results are shown in FIG. 4.
8. Novel washing, denaturation and purification of coronavirus epitope chimeric proteins
The centrifuged pellet (inclusion bodies) was broken up and washed with 250ml of 1mol/L urea prepared with 1 XPBS and 1% Triton-X100 was added thereto, and centrifuged at 8000rpm for 15min at 4℃to obtain a pellet. The pellet after washing the inclusion bodies was redissolved with 200ml of 8mol/L urea, the 8mol/L urea solution was prepared with 1 XPBS, centrifuged again at 8000rpm at 4℃for 15min to collect the supernatant and suction filtered with a 0.45 μm filter. The supernatant after suction filtration was combined with a High Affinity Ni-NTA Affinity chromatography medium previously washed with 1 XPBS several times at 4℃for 1-2 hours with stirring, and then the flow-through peaks were collected via a 12ml plastic chromatography column. Sequentially performing gradient elution with 20, 50, 100, 200 and 500mmol/L imidazole eluent prepared from 8mol/L urea for 15min, collecting eluate under column, and detecting OD 280 Value to OD 280 <0.1 is replaced by imidazole eluent with higher concentration. The flow-through peak and the imidazole eluent (OD 280 The tube with the highest value) was sampled for electrophoresis to determine at which concentration the chimeric protein eluted, and the results are shown in FIG. 5.
9. Dialysis renaturation of novel coronavirus epitope chimeric protein
The purified protein is taken out from the elution peak with high concentration and high purity, and is dialyzed and renatured by 6, 4, 3, 2 and 1mol/L urea and 1 XPBS in sequence, each gradient urea dialysate is prepared by 1 XPBS, the volume of each dialysis is 2L, the dialysis time is 3 hours, glycerol with the final concentration of 10 percent, EDTA with the final concentration of 10mM and DTT with the concentration of 1mM are added in each dialysis, and the renatured chimeric protein is subjected to 12 percent SDS-PAGE electrophoresis detection, so that the yield of the renatured chimeric protein is high. The renatured chimeric protein is preserved at-20 ℃ for standby after adding sterilized glycerol with the final concentration of 20 percent, and the electrophoresis result is shown in figure 6.
Bca method to detect the concentration of novel coronavirus epitope chimeric proteins:
operating according to BCA kit, preparing standard solution, and detecting OD 570 Values, drawing a standard curve, bringing the absorbance value of the chimeric protein into the value, and obtaining the concentration value of the chimeric protein, wherein the standard curve is shown in figure 7.
Results:
1. double enzyme digestion identification of recombinant plasmid:
the extracted recombinant plasmid is digested with EcoR I and HindIII, and lane 2 shows the specific band of 1281bp, which is identical to the expected size, and the recombinant plasmid is constructed successfully, as shown in FIG. 2.
2. Selecting a high expression strain:
lanes 1-6 are 6 single colonies selected, 7-8 are empty plasmid controls, the result shows that all the regulated strains express a large amount of novel coronavirus epitope chimeric protein ENSRBD, and the strain No. 4 with the highest expression level is selected for subsequent expansion culture, and the result is shown in fig. 3.
3. Expression profiling of novel coronavirus epitope chimeric proteins:
the lane 1 shows 1mg/ml BSA, the lane 2 shows the supernatant after disruption, the lane 3 shows the precipitate after disruption, and the electrophoresis result shows that the chimeric protein is mainly expressed in the form of inclusion bodies, and the result is shown in FIG. 4.
4. Purification of novel coronavirus epitope chimeric proteins:
5. Dialysis renaturation of novel coronavirus epitope chimeric proteins:
electrophoresis is carried out on the renatured chimeric protein: lanes 1 and 2-3 are respectively 0.5mg/ml BSA and renatured chimeric proteins, and the results show that the novel soluble coronavirus epitope chimeric proteins are obtained after gradient dialysis renaturation, and the results are shown in FIG. 6.
6. BCA method to detect the concentration of novel coronavirus epitope chimeric proteins:
and (3) drawing a standard curve according to the operation of the BCA kit, bringing the absorbance value of a sample into the curve, and calculating the concentration of the novel coronavirus epitope chimeric protein to be 0.673mg/ml, wherein the standard curve is shown in figure 7.
Preparation and purification of novel coronavirus polyclonal antibody
The purified novel crown fusion Protein ENSRDB is used as an immunogen to be matched with Freund's adjuvant to immunize New Zealand white rabbits, an ear vein blood taking method is adopted after each immunization, the antibody titer and the antibody specificity of rabbit serum are detected, the humoral immune effect induced by the novel crown fusion Protein ENSRDB is evaluated, and the rabbit serum is purified by using a Protein A purification column to obtain the corresponding rabbit polyclonal antibody.
Materials and methods:
1. the novel coronavirus epitope chimeric protein is purified by the preparation of the last part; the Protein A affinity chromatography medium is Nanjing Jinsrui company product; freund's complete adjuvant, freund's incomplete adjuvant, is a product of Sigma company; the TMB color development liquid is a Biyundian company product; the ECL chemiluminescent liquid is a product of Tanon company; the other common reagents are imported split-packs/homemade analytically pure.
2. Preparation of polyclonal antibodies:
(1) Dilution or concentration of novel coronavirus chimeric proteins:
according to the antigen dose arrangement of the rabbit experiment, the amount of chimeric protein required by each rabbit is calculated, and the corresponding dilution or concentration is carried out, wherein the dilution liquid used for dilution is 1 XPBS, and the concentration is carried out by using an ultrafiltration tube with the thickness of 10KD to reach the corresponding volume.
(2) Rabbit immunization protocol:
two New Zealand white rabbits are male, have the weight of about 2kg, are fed by an animal experiment center of an eastern war zone disease control center, are subjected to adaptive culture for a plurality of days, and are immunized according to a pre-plan. Blood is taken from the auricular vein before the first immunization as a negative control, the volume ratio of the chimeric protein solution to Freund's adjuvant is 1:1, and the antigen and the adjuvant are fully mixed and emulsified by vortex oscillation at 4 ℃ in advance. Blood is taken before each immunization to be detected, and the serum is subjected to antibody titer detection after the 4 th immunization so as to determine whether the booster immunization is needed. After the experiment is completed, the experimental rabbits are sacrificed and properly treated, and the specific immunization scheme is as follows:
(3) Specific detection of rabbit serum antibodies:
the specificity of rabbit serum is detected by Western-Blot, novel coronavirus epitope chimeric proteins are subjected to electrophoresis, membrane transfer and sealing are carried out, rabbit serum is used as a primary antibody for incubation, HRP-labeled goat anti-rabbit IgG is used as a secondary antibody for incubation after membrane washing, and the specificity of rabbit serum antibodies is analyzed by membrane washing and development, and the result is shown in figure 8.
(4) Rabbit serum antibody secretion and antiserum titer analysis:
the titers of the rabbit serum after each immunization were detected by ELISA, 96-well plates were coated with novel coronavirus chimeric protein ENSRBD, primary antibody was 10% BSA in 1:1000 dilution of each rabbit antiserum, secondary antibody was 10% BSA in 1:5000 dilution of HRP-labeled goat anti-rabbit IgG, negative control was 10% BSA, and OD was detected using a microplate reader 450 The values, which give a change in serum antibody secretion with increasing number of immunizations, are shown in FIG. 9. ELISA is adopted to detect the titer of antiserum after the fourth immunization, a novel coronavirus chimeric protein ENSRBD is used for coating a 96-well plate, the primary antibody is the antiserum after the fourth immunization which is diluted in a gradient way, the secondary antibody is HRP-marked goat anti-rabbit IgG, and an enzyme-labeled instrument is used for detecting OD 450 The value of the four-immune post-rabbit anti-blood is obtainedThe results of the titers of the supernatants are shown in FIG. 10.
3. Purification of polyclonal antibodies:
the rabbit blood was left to settle naturally after 1h at room temperature, then left to separate in 4℃for 4h, and then centrifuged at 12000rpm for 15min, and the supernatant serum was aspirated in a sterile console.
(1) Sample preparation: the negative serum and the serum taken after 4 times of immunization are respectively diluted by balancing solution, wherein the dilution ratio is at least 1:1, so that the antiserum is ensured to have certain ionic strength and PH value so as to be better combined with Protein A medium.
(2) And (3) column loading: and (3) fully shaking the Protein A uniformly, then sucking and transferring the Protein A into a 10ml chromatographic column for a small amount of times by using a pipetting gun, opening a liquid outlet to allow the preservation liquid to flow out, naturally settling the medium, closing a liquid outlet valve after the medium is settled completely, sucking 5-10 ml balance liquid balance colloid for 15min, and opening the liquid outlet again to allow the balance liquid to flow out.
(3) Medium binding to sample: the equilibrated medium was mixed with sample serum and then combined overnight at 4℃and the next day on the column and the flow-through peaks were collected.
(4) Washing: firstly, balancing colloid again by using a balancing solution, detecting the absorbance value of effluent liquid in real time, and waiting for the absorbance value A 280 After stabilization, purification was performed.
(5) Purifying: adding small amount of eluent into the chromatographic column, regulating flow rate of the effluent, detecting absorbance value of the effluent in real time, and waiting for A 280 When the pH value is too low, the elution is stopped, 1ml of the neutralization buffer is immediately sucked up and added to the chromatographic column, and the pH value is adjusted to 7.4.
(6) Electrophoresis: the 4 serum purified by Protein A were each sampled for gel electrophoresis, and the results are shown in FIG. 11.
Results:
1. specificity of rabbit serum antibodies:
the specificity of rabbit serum is detected by Western-Blot, and lane 1 is novel coronavirus epitope chimeric protein, and the result shows that the specificity of rabbit serum antibody is better, as shown in figure 8.
2. Secretion of rabbit serum antibodies:
ELISA was used to detect the titer of rabbit serum after each immunization, and serum antibody OD was increased with increasing number of immunizations 450 The value is also increasing, and the antibody OD is obtained in the fourth immune serum 450 The value reached the highest and the result is shown in figure 9.
3. Rabbit serum antibody titers:
the titers of antisera after the fourth immunization were measured by ELISA, and as can be seen from FIG. 10, the titers of rabbit serum antibodies after the fourth immunization reached 1:1280000.
4. Purification of polyclonal antibodies:
the results of gel electrophoresis of the 4 serum samples purified by Protein A column were shown in FIG. 11, which shows that the concentration of polyclonal antibody was continuously increased with increasing number of immunizations and reached the highest after the fourth immunization.
Establishment and evaluation of novel coronavirus double-antibody sandwich ELISA detection method
A double-antibody sandwich ELISA detection method for detecting a novel coronavirus antigen is established by taking a rabbit-derived polyclonal antibody as a capture antibody and taking an HRP-marked rabbit-derived polyclonal antibody as an enzyme-labeled antibody. The dilution degree, the type and the blocking time of the optimal capture antibody and the enzyme-labeled antibody are respectively determined, the minimum detection limit and the specificity of the established ELISA method are preliminarily evaluated, the influence of different sample storage conditions on the minimum detection limit of the ELISA method is compared, and the established double-antibody sandwich ELISA method is hopefully used for detecting the novel coronavirus antigen.
Materials and methods:
1. novel coronavirus polyclonal antibodies are prepared as the last part; the general human adenovirus epitope chimeric protein, mycoplasma pneumoniae P1C, rubella virus 2 XRub, respiratory syncytial virus RSV, parainfluenza virus HN3 and other antigen proteins are prepared and stored in the laboratory; the HRP enzyme-labeled goat anti-rabbit IgG is a Borson company product; the TMB color development liquid is a Biyundian company product; the ECL chemiluminescent liquid is a product of Tanon company; the other common reagents are imported split-packs/homemade analytically pure.
2. Establishment and evaluation of a double antibody sandwich ELISA detection method:
(1) Primary treatment of polyclonal antibody:
the purified rabbit polyclonal antibody was dialyzed against 1 XPBS, 2L each time, 5 times at 3h intervals to reduce the Tris concentration in the polyclonal solution and remove glycine and amine. And regulating and detecting the pH value of the polyclonal antibody after dialysis to reach a proper range of 6.5-8.5. The concentration of the polyclonal antibody after dialysis was measured by BCA method, the polyclonal antibody was diluted to 1. Mu.g/. Mu.L with 1 XPBS, and the polyclonal antibody was placed at-20℃for use after sub-packaging, and the standard curve is shown in FIG. 12.
(2) Preparation of enzyme-labeled polyclonal antibody:
adding 100 mu L of the dialyzed polyclonal antibody solution into a centrifuge tube which is pre-filled with 10 mu L Modifier reagent, lightly blowing and mixing the solution with a pipetting gun for several times, adding the solution into a centrifuge tube which contains 100 mu g of HRP-mix powder, blowing and mixing the solution again, incubating the solution for 3 hours at room temperature in a dark place, adding 10 mu L Quencher reagent into the tube, and standing for 30min to obtain the product.
(3) Determination of optimal working concentration of capture antibody and detection antibody:
the purified rabbit polyclonal antibody is used as a capture antibody, CBS coating liquid is used for double dilution (1:200-1:25600), novel coronavirus epitope chimeric protein is used as an antigen, PBST is used as a negative control, a detection antibody is double dilution (1:200-1:25600) HRP marked rabbit polyclonal antibody, and OD is measured by an enzyme-labeled instrument after color development 450 The values were calculated and the P/N values were calculated to give the optimal concentration combinations of capture and detection antibodies, the results are shown in table 1.
TABLE 1 determination of optimal capture antibody and detection antibody dilution combination
(4) Selection of the optimal type of blocking solution:
three blocking solutions were set, 3% BSA, 5% BSA and 5% skimmed milk powder, respectivelyThe rabbit-derived polyclonal antibody with the optimal dilution is used as a capture antibody, the HRP-labeled polyclonal antibody with the optimal dilution is used as a detection antibody, the purified novel coronavirus chimeric protein ENSRBD is used as an antigen, a compound well and a PBST negative control are arranged, and after color development, the OD is measured by an enzyme-labeled instrument 450 The values were calculated and P/N values were calculated to give the best confining liquid species, the results are shown in Table 2.
TABLE 2 determination of optimal confining liquid species
(5) Determination of optimal closing time:
setting 6 blocking times of 0.5h, 1h, 1.5h, 2h, 2.5h and 3h respectively, detecting new coronal fusion protein by adopting optimal capture antibody, detection antibody dilution combination and optimal blocking solution, taking purified new coronal virus chimeric protein ENSRBD as antigen, setting a compound hole and PBST negative control, and measuring OD by an enzyme-labeled instrument after color development 450 The values were calculated and the P/N values were calculated to give the best blocking time and the results are shown in Table 3.
TABLE 3 determination of optimal closing time
the initial concentration of the purified novel coronavirus epitope chimeric protein ENSRBD is 0.5mg/mL, and the following steps are sequentially carried out according to 1: 25. 1:50, 1: 100. dilution was performed at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, the dilutions were 1 XPBS, ELISA detection was performed with the combination of optimal capture antibody and detection antibody dilutions, optimal blocking liquid species, optimal blocking time, and the minimum detection limit and linear range of the ELISA method were obtained, and the results are shown in Table 4, FIG. 13.
TABLE 4 determination of minimum detection limit of double antibody sandwich ELISA
(7) Specificity of the double antibody sandwich ELISA detection method:
according to the ELISA detection method established in the process, other antigens such as novel coronavirus ENSRBD protein, parainfluenza virus HN3, respiratory syncytial virus RSV, mycoplasma pneumoniae P1C, rubella virus 2 xRub, human adenovirus HAdV and the like are respectively detected, and the detection results are judged according to the P/N value so as to evaluate the specificity of the established ELISA detection method, and the result is shown in figure 14. The specificity calculation method here is as follows: specificity = number of antigen types that the method detects as negative/number of antigen types that are actually negative.
(8) Effect of sample storage conditions on method detection limits:
three room temperature times were set: 25 ℃ for 1h, 4h and 8h; three freeze thawing times are set: repeatedly freezing and thawing for 1 time, 3 times and 5 times. The initial concentration of the purified novel coronavirus ENSRBD protein is 0.5mg/mL, and the concentration is sequentially 1: 25. 1:50, 1: 100. dilution was performed at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200. ELISA detection is carried out by combining the optimal capture antibody and the concentration of the detection antibody, the optimal blocking liquid type and the optimal blocking time, 3% BSA and PBST are used as negative controls, P/N values of all holes are calculated, the lowest detection limit of an ELISA method under various sample storage conditions is obtained, the influence of the sample storage conditions on the detection method is analyzed, and the results are shown in tables 5-6.
TABLE 5 influence of sample room temperature standing time on method minimum detection limit
TABLE 6 influence of repeated freeze thawing times of samples on the minimum detection limit of the method
Results:
1. determination of optimal working concentration of capture antibody and detection antibody:
determining the optimal dilution combination of capture antibody and detection antibody using a square method is: the capture antibody dilution was 1:6400 and the detection antibody dilution was 1:800, the results are shown in Table 1.
2. Selection of the optimal type of blocking solution:
Three sealing liquids are arranged: the blocking time was 2h for 3% BSA, 5% BSA and 5% nonfat dry milk, and the best blocking solution species was 3% BSA from the maximum P/N values, and the results are shown in Table 2.
3. Determination of optimal closing time:
setting 6 closing times: the blocking solutions were 3% BSA at 0.5h, 1h, 1.5h, 2h, 2.5h, and 3h, and the optimal blocking time was 2h from the maximum P/N value, and the results are shown in Table 3.
4. Minimum limit of detection and linear range of double antibody sandwich ELISA detection method:
the initial concentration of novel coronavirus epitope chimeric protein ENSRBD is 0.5mg/mL according to 1: gradient dilution is carried out at 25-1:51200, the minimum detection limit of the method is 0.039 ng/. Mu.L according to the maximum P/N value, and OD is calculated 570 The values and antigen concentration values were logarithmic, giving a linear range of 0.039-1.25 ng/. Mu.L for this ELISA method, results are shown in Table 4, FIG. 13.
5. Specificity of the double antibody sandwich ELISA detection method:
the established ELISA detection method is used for detecting novel coronavirus ENSRBD protein as a positive result, and detecting parainfluenza virus HN3, respiratory syncytial virus RSV, mycoplasma pneumoniae P1C, rubella virus 2 xRub, human adenovirus HAdV and other antigens as negative results, and the result shows that the established ELISA detection method has better specificity (the specificity = the number of antigen types of which the detection result is negative/the number of antigen types of which the detection result is actually negative), and the figure 14 is shown.
6. Effect of sample storage conditions on method detection limits:
three room temperature times were set: 25 ℃ for 1h, 4h and 8h; three freeze thawing times are set: repeated freezing and thawing for 1 time, 3 times and 5 times, detecting the novel coronavirus ENSRBD protein subjected to gradient dilution by using an established ELISA method, wherein the minimum detection limits at 25 ℃ for 1h, 4h and 8h are respectively 0.156 ng/mu L, 0.625 ng/mu L and show a trend of increasing along with the extension of the room temperature standing time; the minimum detection limits of the repeated freezing and thawing for 1 time, 3 times and 5 times are 0.156 ng/mu L, 0.156 ng/mu L and 0.313 ng/mu L respectively, and the repeated freezing and thawing tends to increase with the increase of the repeated freezing and thawing times; the results are shown in tables 5-6.
Preparation of novel coronavirus polyclonal antibody and application sequence table thereof are shown in the accessory document: a nucleotide or amino acid sequence listing computer readable carrier.
Claims (7)
1. The preparation and application of a novel coronavirus polyclonal antibody relate to a novel coronavirus antigen epitope chimeric protein, the chimeric protein is formed by connecting an extracellular region segment of a novel coronavirus envelope protein E, an N-terminal segment of a nucleocapsid protein N and a receptor binding domain RBD segment of spike protein S, the protein segments are connected by two glycine and one serine, the chimeric protein has the total length of 422 amino acids, and the amino acid sequence is as follows:
2. A novel coronavirus epitope chimeric protein coding gene according to claim 1, wherein: the nucleotide sequence is as follows:
ATG TAT AGT TTT GTA TCA GAA GAG ACA GGA ACT CTG ATC GTA AAT TCC GGT GGT AGCATG AGC GAC AAC GGC CCT CAA AAT CAA CGT AAC GCT CCG CGT ATT ACC TTC GGT GGC CCG TCT GAC TCC ACA GGC AGC AAC CAG AAT GGT GAA CGT AGC GGT GCACGT AGC AAA CAG CGT CGC CCT CAG GGC CTG CCG AAC AAC ACC GCT AGC TGG TTT ACC GCC TTG ACC CAG CAC GGC AAA GAA GAT CTG AAG TTT CCG CGA GGT CAG GGC GTG CCG ATT AAC ACC AAC TCT AGC CCG GAC GAC CAG ATC GGT TAT TAC CGT CGT GCT ACC CGT CGC ATC AGA GGC GGG GAT GGT AAG ATG AAA GAC TTA AGC CCG CGT TGG TAT TTT TAC TAT CTG GGT ACT GGT CCA GAG GCA GGT CTG CCG TAC GGC GCG AAC AAG GAT GGG ATT ATC TGG GTT GCG ACG GAA GGA GCG CTG AAT ACT CCG AAG GAT CAT ATC GGT ACG CGT AAT CCG GCT AAC AAT GCC GCA ATC GTG CTC CAA CTG CCA CAG GGT ACC ACC CTG CCG AAG GGT TTT TAT GCC GAG GGT AGC CGC GGC GGT TCA CGT GTC CAG CCG ACC GAG AGC ATT GTT CGC TTT CCG AAC ATT ACG AAC CTT TGC CCG TTC GGT GAG GTG TTC AAC GCG ACC CGT TTC GCA AGC GTT TAC GCG TGG AAC CGT AAG CGC ATT TCT AAC TGC GTG GCG GAC TAC AGC GTG CTG TAC AAC AGC GCT TCG TTC AGC ACG TTT AAA TGT TAC GGT GTG TCC CCG ACC AAA TTG AAC GAT CTG TGT TTC ACC AAT GTT TAC GCC GAC TCG TTT GTT ATC CGT GGT GAC GAA GTT CGT CAG ATT GCG CCA GGT CAA ACC GGT AAG ATC GCA GAC TAT AAC TAC AAA CTG CCA GAT GAT TTT ACT GGC TGC GTC ATC GCG TGG AAC AGC AAC AAT CTA GAT AGT AAG GTT GGC GGT AAC TAC AAC TAC TTG TAT CGT CTG TTC CGC AAG AGC AAT CTG AAA CCG TTC GAG CGC GAC ATC TCT ACC GAA ATT TAT CAA GCG GGC TCT ACC CCG TGC AAT GGC GTG GAG GGC TTC AAT TGC TAC TTC CCG TTA CAA TCC TAT GGC TTC CAA CCG ACC AAC GGT GTG GGC TAC CAG CCG TAT CGC GTG GTT GTT CTG TCC TTT GAA CTG CTG CAC GCG CCG GCG ACC GTT TGT GGC CCG AAA AAA TCT ACC AAC TTG GTG AAA AAC AAG TGC GTC AAT TTC TAA。
3. the preparation and application of the novel coronavirus polyclonal antibody use escherichia coli to express novel coronavirus antigen epitope chimeric protein, and the specific preparation method is as follows:
(3.1) construction of SARS-CoV-2 epitope chimeric protein recombinant plasmid:
extracting plasmid pET-28a (+), double enzyme cutting with EcoRI and SalI, electrophoresis, recovering large fragment of enzyme cut plasmid, dissolving in deionized water, double enzyme cutting with EcoRI and SalI to synthesize SARS-CoV-2 antigen epitope chimeric protein gene fragment, electrophoresis recovering, dissolving in deionized water, taking the DNA fragment after enzyme cutting, inserting between EcoRI and SalI sites in carrier pET-28a (+), expressing chimeric protein containing multiple novel coronavirus antigen epitopes.
(3.2) screening and identification of recombinant plasmids:
transforming the recombinant plasmid into escherichia coli BL21 (DE 3), coating an LB plate containing kanamycin, standing overnight at 37 ℃, randomly picking a transformed colony the next day to extract the plasmid, and carrying out double enzyme digestion verification by EcoRI and HindIII, wherein a 1.0% agarose gel electrophoresis result shows that a 1281bp target gene fragment is cut off; meanwhile, the plasmid containing the gene fragment is subjected to DNA sequence analysis, and the sequence analysis proves that the sequence of the recombinant plasmid is completely correct:
ATG TAT AGT TTT GTA TCA GAA GAG ACA GGA ACT CTG ATC GTA AAT TCC GGT GGT AGC ATG AGC GAC AAC GGC CCT CAA AAT CAA CGT AAC GCT CCG CGT ATT ACC TTC GGT GGC CCG TCT GAC TCC ACA GGC AGC AAC CAG AAT GGT GAA CGT AGC GGT GCA CGT AGC AAA CAG CGT CGC CCT CAG GGC CTG CCG AAC AAC ACC GCT AGC TGG TTT ACC GCC TTG ACC CAG CAC GGC AAA GAA GAT CTG AAG TTT CCG CGA GGT CAG GGC GTG CCG ATT AAC ACC AAC TCT AGC CCG GAC GAC CAG ATC GGT TAT TAC CGT CGT GCT ACC CGT CGC ATC AGA GGC GGG GAT GGT AAG ATG AAA GAC TTA AGC CCG CGT TGG TAT TTT TAC TAT CTG GGT ACT GGT CCA GAG GCA GGT CTG CCG TAC GGC GCG AAC AAG GAT GGG ATT ATC TGG GTT GCG ACG GAA GGA GCG CTG AAT ACT CCG AAG GAT CAT ATC GGT ACG CGT AAT CCG GCT AAC AAT GCC GCA ATC GTG CTC CAA CTG CCA CAG GGT ACC ACC CTG CCG AAG GGT TTT TAT GCC GAG GGT AGC CGC GGC GGT TCA CGT GTC CAG CCG ACC GAG AGC ATT GTT CGC TTT CCG AAC ATT ACG AAC CTT TGC CCG TTC GGT GAG GTG TTC AAC GCG ACC CGT TTC GCA AGC GTT TAC GCG TGG AAC CGT AAG CGC ATT TCT AAC TGC GTG GCG GAC TAC AGC GTG CTG TAC AAC AGC GCT TCG TTC AGC ACG TTT AAA TGT TAC GGT GTG TCC CCG ACC AAA TTG AAC GAT CTG TGT TTC ACC AAT GTT TAC GCC GAC TCG TTT GTT ATC CGT GGT GAC GAA GTT CGT CAG ATT GCG CCA GGT CAA ACC GGT AAG ATC GCA GAC TAT AAC TAC AAA CTG CCA GAT GAT TTT ACT GGC TGC GTC ATC GCG TGG AAC AGC AAC AAT CTA GAT AGT AAG GTT GGC GGT AAC TAC AAC TAC TTG TAT CGT CTG TTC CGC AAG AGC AAT CTG AAA CCG TTC GAG CGC GAC ATC TCT ACC GAA ATT TAT CAA GCG GGC TCT ACC CCG TGC AAT GGC GTG GAG GGC TTC AAT TGC TAC TTC CCG TTA CAA TCC TAT GGC TTC CAA CCG ACC AAC GGT GTG GGC TAC CAG CCG TAT CGC GTG GTT GTT CTG TCC TTT GAA CTG CTG CAC GCG CCG GCG ACC GTT TGT GGC CCG AAA AAA TCT ACC AAC TTG GTG AAA AAC AAG TGC GTC AAT TTC TAA
(3.3) screening and identifying engineering bacteria expressing chimeric proteins:
inoculating positive transformant containing recombinant plasmid into a test tube containing 4mLLB culture medium, the culture medium contains kana toxin 50 mug/mL, shake culturing overnight at 37 ℃, adding IPTG to a final concentration of 0.2mmol/L the next day, continuing shake culturing and inducing for 4 hours, centrifuging to collect thalli for SDS-PAGE detection, expressing SARS-CoV-2 epitope chimeric protein with relative molecular weight of 46.52kD, and selecting a strain with highest chimeric protein expression amount for subsequent experiments in the absence of this protein band of control bacterium BL21 (DE 3).
(3.4) purification of SARS-CoV-2 epitope chimeric protein:
(3.4.1) ultrasonic cleavage of SARS-CoV-2 epitope chimeric protein engineering bacterium:
carrying out induced expression on the high-expression recombinant bacteria after inoculating, expanding and storing strains, centrifuging engineering bacteria for inducing and expressing chimeric proteins to obtain bacteria, wherein the centrifugation condition is 8000rpm, 15min and 4 ℃, preparing bacterial lysate with 1/10 volume of an original culture solution, wherein the lysate contains 20mmol/L PB,5% glycerol and the pH value is 7.4; adding lysozyme and Leupeptin, pepstatin, pmsf, mixing, and suspending the centrifuged strain. The conditions of ice bath ultrasonic breaking are breaking time 4s, interval time 4s and breaking 75 times, centrifuging again, collecting supernatant and sediment, respectively reserving samples, and detecting the expression form of the chimeric protein by 12% SDS-PAGE electrophoresis, and the electrophoresis result shows that the chimeric protein is mainly expressed in the form of inclusion bodies.
(3.4.2) inclusion body washing and denaturation:
the inclusion bodies were redissolved with 200mL of 1mol/L urea prepared with 1 XPBS in a urea solution of 1 XPBS, 1% Triton X-100 was added, washed and centrifuged at 8000rpm at 4℃for 15min to leave a precipitate, the precipitate was denatured with 200mL of 8mol/L urea, and the urea solution was centrifuged at 8000rpm at 4℃for 15min to collect the supernatant.
(3.4.3) chimeric proteins bind to cement:
to a 12mL plastic column, 5mL of High-Affinity Ni-NTA Resin was added, and the gel was washed 5 times with 1 XPBS to remove residual ethanol in the gel. The supernatant from step 3.4.2 was filtered through a 0.45 μm filter paper and combined with the prepared gum under stirring in an ice bath for 1-2h.
(3.4.4) elution of chimeric proteins:
collecting the flow-through peaks of the mixture of the protein and the gel through a plastic chromatographic column, fully washing the column by using 8mol/L urea as a balance liquid, eluting the protein by sequentially using 20, 50, 100, 200 and 500mmol/L imidazole, preparing the imidazole solution by using 8mol/L urea, eluting for 15min each time, collecting the proteins of each eluting peak, and detecting the OD of the proteins 280 Value up to protein OD 280 Below 0.1, the imidazole eluent is changed into an imidazole eluent with higher concentration. Each elution peak protein was detected by 12% SDS-PAGE electrophoresis to determine which elution peak contained the expressed SARS-CoV-2 epitope chimeric protein. Electrophoresis results show that 20 mmol/L, 50 mmol/L and 100mmol/L imidazole elution protein peaks contain novel coronavirus epitope chimeric proteins.
4. The preparation and application of the novel coronavirus polyclonal antibody use the novel coronavirus epitope chimeric protein as immunogen, the novel coronavirus epitope chimeric protein is matched with Freund's adjuvant to immunize New Zealand white rabbits, rabbit serum is taken for purification, and the corresponding rabbit-derived polyclonal antibody is prepared, and the polyclonal antibody is characterized in that the novel coronavirus epitope chimeric protein can be identified, and the preparation and purification methods of the polyclonal antibody are as follows:
(4.1) preparation of novel coronavirus polyclonal antibodies:
taking novel coronavirus epitope chimeric protein as immunogen, immunizing rabbits with Freund's adjuvant at 500 mug each, performing whole-course immunization according to a pre-designed scheme, detecting specificity of antiserum by Western-Blot, and taking blood to detect antibody OD after each immunization 450 The value, analyze the secretion situation of serum antibody with increasing the number of times of immunization, carry on the gradient dilution to the serum after the fourth time of immunization, detect the potency of the antiserum through ELISA method, the result shows that the specificity of the antibody of rabbit serum is better; OD of rabbit serum antibody with increasing immunization times 450 The value is also increasing, and the titer of serum antibodies reaches the maximum after the fourth immunization, the titer is 1:1280000;
(4.2) purification of novel coronavirus polyclonal antibodies:
separating and purifying polyclonal antibody from serum after four times immunization by Protein A column affinity chromatography, standing the blood at room temperature for 1h after each blood taking, standing for layering at 4 ℃ for 4h, centrifuging at 12000rpm for 15min, sucking upper serum for standby in a sterile operation table, dialyzing the serum by 1 XPBS, diluting with dialysate, mixing the diluted serum with medium overnight, and changing the affinity of the medium and the antibody by changing the pH value of the solution, thereby eluting the antibody; the 4 immune sera purified by Protein A column were sampled separately for gel electrophoresis analysis. As shown by the electrophoresis result, the concentration of the polyclonal antibody continuously increases with the increase of the immunization times, and reaches the highest value after the fourth immunization, and the concentration of the polyclonal antibody reaches 1.34mg/mL as measured by the BCA method.
5. The method for establishing double-antibody sandwich ELISA by using the novel coronavirus rabbit-derived polyclonal antibody prepared in accordance with claim 4, and evaluating the minimum detection limit and the specificity of the ELISA method comprises the following specific steps:
(1) Dialyzing the purified rabbit polyclonal antibody with 1 XPBS, detecting pH value of the polyclonal antibody after dialysis to reach a proper range of 6.5-8.5, diluting the polyclonal antibody to 1 mug/mu L with 1 XPBS, subpackaging, and standing at-20deg.C for use;
(2) Preparing HRP-labeled polyclonal antibody from the dialyzed polyclonal antibody solution by using an HRP-labeled antibody kit;
(3) The purified rabbit polyclonal antibody is used as a capture antibody, the HRP-labeled rabbit polyclonal antibody is used as a detection antibody, and the optimal dilution combination of the capture antibody and the detection antibody is obtained by a square method, wherein the dilution combination is as follows: the dilution of the capture antibody was 1:6400 and the dilution of the detection antibody was 1:800;
(4) Three sealing liquids are arranged: 3% BSA, 5% BSA and 5% skim milk powder, and detection was performed using the ELISA method, and the optimal blocking solution type was determined to be 3% BSA based on the P/N value;
(5) Six closing times are set: 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, using the ELISA method for detection, determining the optimal closing time to be 2h according to the P/N value;
(6) The initial concentration of novel coronavirus epitope chimeric protein ENSRBD is 0.5mg/mL according to 1: diluting 25-1:51200, and obtaining the lowest detection limit of the ELISA method to be 0.039 ng/. Mu.L according to the P/N value of > 2.1; taking the logarithm of the absorbance value and the chimeric protein concentration value respectively to obtain the linear range of 0.039-1.25 ng/. Mu.L of the ELISA method;
(7) The established ELISA detection method is used for respectively detecting novel coronavirus ENSRBD proteins and other common respiratory tract pathogen antigens, judgment is carried out according to P/N values, the detection result shows that other pathogen antigens are negative except that the novel coronavirus is positive, and the established ELISA detection method has good specificity;
(8) Effect of sample storage conditions on detection limit of ELISA method:
three room temperature times were set: 25 ℃ for 1h, 4h and 8h; three freeze thawing times are set: repeatedly freezing and thawing for 1 time, 3 times and 5 times; the established ELISA method is used for detecting the new crown chimeric protein diluted in a gradient way, the influence of the sample storage condition on the detection limit of the method is analyzed, and the result shows that the detection limit of the ELISA method is improved along with the extension of the room temperature standing time and the increase of the repeated freezing and thawing times, so that the ELISA method is used for avoiding the long-time room temperature standing and repeated freezing and thawing of the sample.
6. The polyclonal antibody corresponding to the novel coronavirus epitope chimeric protein of claim 4, which is used for detecting SARS-CoV-2 antibody or antigen.
7. The application of the novel coronavirus double-antibody sandwich ELISA detection method in the field of novel coronavirus antigen detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210915208.0A CN116284431A (en) | 2022-08-01 | 2022-08-01 | Preparation and application of novel coronavirus polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210915208.0A CN116284431A (en) | 2022-08-01 | 2022-08-01 | Preparation and application of novel coronavirus polyclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116284431A true CN116284431A (en) | 2023-06-23 |
Family
ID=86820890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210915208.0A Pending CN116284431A (en) | 2022-08-01 | 2022-08-01 | Preparation and application of novel coronavirus polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116284431A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117330750A (en) * | 2023-12-01 | 2024-01-02 | 北京生物制品研究所有限责任公司 | Method for screening early virus seed of new coronavirus and method for preparing vaccine |
-
2022
- 2022-08-01 CN CN202210915208.0A patent/CN116284431A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117330750A (en) * | 2023-12-01 | 2024-01-02 | 北京生物制品研究所有限责任公司 | Method for screening early virus seed of new coronavirus and method for preparing vaccine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| GB2604692A (en) | Method for preparing recombinant subunit vaccine against novel coronavirus | |
| CN109970851B (en) | Monoclonal antibody of CCV virus M protein, preparation method thereof and preparation method of immune colloidal gold test strip | |
| CN110845582B (en) | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof | |
| CN116284431A (en) | Preparation and application of novel coronavirus polyclonal antibody | |
| CN113203854B (en) | Indirect ELISA kit for detecting cat coronavirus antibody | |
| CN112125961B (en) | Bovine viral diarrhea-bovine infectious rhinotracheitis bigeminal subunit fusion vaccine and identification method thereof | |
| CN107167609B (en) | Distinguish the antibody indirect ELISA detection method of pig blue-ear disease street strain and the strain of vaccine strain Tianjin | |
| CN113150126A (en) | Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof | |
| CN110257405B (en) | Mycoplasma bovis alcohol dehydrogenase gene and encoding protein and application thereof | |
| CN110687302A (en) | Test paper strip and method for rapidly detecting porcine circovirus type 2 antigen or antibody colloidal gold | |
| CN111621506A (en) | Mycoplasma bovis secretory protein Mbovp0145 and application thereof | |
| CN111499697A (en) | Indirect E L ISA antibody detection kit for African swine fever virus p54 recombinant protein and preparation method thereof | |
| CN113980908B (en) | Actinobacillus pleuropneumoniae ApxIV protein monoclonal antibody and blocking ELISA kit thereof | |
| CN116240222A (en) | Codon-optimized bovine viral diarrhea virus type 1E2 protein gene and application thereof | |
| CN115772230A (en) | RSV (respiratory syncytial virus) recombinant antigen as well as preparation method and application thereof | |
| KR101080071B1 (en) | Rift valley fever competition ELISA using monoclonal antibodies against recombinant N protein | |
| KR20230123463A (en) | Recombinant classical swine fever virus E2 protein | |
| CN110554187B (en) | Expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof | |
| CN111393513A (en) | Mycoplasma bovis secretory adhesive protein MbovP581 | |
| CN113512098B (en) | Indirect ELISA (enzyme-Linked immuno sorbent assay) method for identifying swine fever virus and bovine viral diarrhea virus serum antibodies and application thereof | |
| CN116041541B (en) | Mycobacterium tuberculosis antigen EPPA011 and application thereof | |
| CN116121197B (en) | Monoclonal antibody of anti-iridovirus SDDV isolate of yellow-fin sea bream and application thereof | |
| CN117143207B (en) | Recombinant epitope protein, encoding gene, recombinant plasmid, preparation method, engineering bacteria and application | |
| CN110967483B (en) | Detection kit for malignant catarrhal fever virus, preparation method and application thereof | |
| CN117886896B (en) | African swine fever multi-epitope recombinant protein ASFVMEA and application |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230911 Address after: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Applicant after: EASTERN THEATER DISEASE PREVENTION AND CONTROL CENTER OF PLA Address before: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Applicant before: Li Yuexi |
|
| TA01 | Transfer of patent application right |