CN116903733A - Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus - Google Patents
Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus Download PDFInfo
- Publication number
- CN116903733A CN116903733A CN202311167339.6A CN202311167339A CN116903733A CN 116903733 A CN116903733 A CN 116903733A CN 202311167339 A CN202311167339 A CN 202311167339A CN 116903733 A CN116903733 A CN 116903733A
- Authority
- CN
- China
- Prior art keywords
- phage
- nanobody
- influenza
- antibody
- nano antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 206010022000 influenza Diseases 0.000 title claims abstract description 34
- 101710154606 Hemagglutinin Proteins 0.000 title claims abstract description 28
- 101710093908 Outer capsid protein VP4 Proteins 0.000 title claims abstract description 28
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 title claims abstract description 28
- 101710176177 Protein A56 Proteins 0.000 title claims abstract description 28
- 239000000185 hemagglutinin Substances 0.000 title claims abstract description 27
- 238000012216 screening Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 21
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 230000009385 viral infection Effects 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 73
- 239000007788 liquid Substances 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 239000006228 supernatant Substances 0.000 claims description 32
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 30
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 30
- 239000003480 eluent Substances 0.000 claims description 28
- 238000002965 ELISA Methods 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- 238000002823 phage display Methods 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 17
- 239000012895 dilution Substances 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 13
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- 241000282836 Camelus dromedarius Species 0.000 claims description 11
- 238000010276 construction Methods 0.000 claims description 11
- 241001416177 Vicugna pacos Species 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 241001052560 Thallis Species 0.000 claims description 8
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000012423 maintenance Methods 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 5
- 230000035755 proliferation Effects 0.000 claims description 5
- 238000010839 reverse transcription Methods 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 108700010900 influenza virus proteins Proteins 0.000 claims description 4
- 238000001784 detoxification Methods 0.000 claims description 3
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 241001515965 unidentified phage Species 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 description 22
- 239000000243 solution Substances 0.000 description 20
- 241000700605 Viruses Species 0.000 description 16
- 102000036639 antigens Human genes 0.000 description 14
- 108091007433 antigens Proteins 0.000 description 14
- 238000003776 cleavage reaction Methods 0.000 description 14
- 230000007017 scission Effects 0.000 description 13
- 239000002609 medium Substances 0.000 description 11
- 239000000427 antigen Substances 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 241000287828 Gallus gallus Species 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 210000000170 cell membrane Anatomy 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 108010006232 Neuraminidase Proteins 0.000 description 7
- 102000005348 Neuraminidase Human genes 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 230000003248 secreting effect Effects 0.000 description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229910009891 LiAc Inorganic materials 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 241000271566 Aves Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000010530 Virus Neutralization Effects 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000004091 panning Methods 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241000712431 Influenza A virus Species 0.000 description 2
- 241000712464 Orthomyxoviridae Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 102100040646 Fc receptor-like B Human genes 0.000 description 1
- 101710142642 Fc receptor-like B Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101710114810 Glycoprotein Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101100113485 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) chs-3 gene Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 244000253724 Saccharomyces cerevisiae S288c Species 0.000 description 1
- 235000004905 Saccharomyces cerevisiae S288c Nutrition 0.000 description 1
- 101001039853 Sonchus yellow net virus Matrix protein Proteins 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010042566 Superinfection Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 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
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
-
- 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
- G01N2333/11—Orthomyxoviridae, e.g. influenza 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Plant Pathology (AREA)
Abstract
The invention provides an anti-influenza H9 hemagglutinin nano antibody and an antibody screening method for cells infected by using influenza viruses, wherein the anti-influenza H9 hemagglutinin nano antibody is a nano antibody L1-2, a nano antibody L1-4 or a nano antibody A-5, the amino acid sequence of the nano antibody L1-2 is shown as SEQ ID NO.16, the amino acid sequence of the nano antibody L1-4 is shown as SEQ ID NO.17, and the amino acid sequence of the nano antibody A-5 is shown as SEQ ID NO. 18. The invention also provides application of the anti-influenza H9 hemagglutinin nano-antibody, and application of the anti-influenza H9 hemagglutinin nano-antibody in preparation of a medicament for treating influenza virus infection; the anti-influenza H9 hemagglutinin nano antibody is applied to preparation of medicaments, reagents, detection plates or kits for detecting influenza virus infection.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an anti-influenza H9 hemagglutinin nano antibody and an antibody screening method for infected cells by using influenza viruses.
Background
In 1993, belgium immunologists Hamers Casterman et al reported for the first time in Nature that in camel serum there was a Heavy chain antibody (HCAb) that naturally lacks the light chain and Constant region CH1 (Constant region 1) in addition to the conventional tetrameric antibody IgG 1. The HCAbs variable region of camelids can independently form a complete antigen binding site, designated as heavy chain variable region single domain antibody (VHH), also known as Nanobody (Nb). Nanobody molecular weight is about 15 kDa, only one tenth of that of ordinary antibodies, being the smallest fragment known to date to bind antigen. The nano antibody has the characteristics of high affinity, high specificity, stability, high tissue penetrability, low immunogenicity, easy gene modification and low production cost, and has good application prospect in the field of antiviral treatment. The nano antibody is taken as an ideal element for developing multiple structural domains, and can be connected with the nano antibody or other proteins in series to form a novel fusion molecule by a genetic engineering technology, so that the novel fusion molecule has the antigen binding activity of the nano antibody and the functions of other proteins. For example, the half-life of the nanobody is short, the blood clearance rate is high, the single-chain antibody can be constructed by fusion expression with the IgY Fc region of chicken, and the VHH-IgY Fc is prevented from being cracked by lysosomes by utilizing the characteristic that the Fc domain can be combined with an IgY receptor (FcRY), so that the half-life of the nanobody in vivo is prolonged.
In 1985, smith first reported phage display technology. After 20 years of development perfection, phage display technology has been widely applied to antibody library establishment, drug design, vaccine research, pathogen detection, epitope research, cell signal transduction research and the like. Because the gene for encoding the exogenous protein is inserted into the phage capsid protein gene, the gene is fused and expressed with the capsid protein, and the phage display technology establishes the corresponding relation between genotype and phenotype. The monovalent phagemid display system comprising a phagemid vector and an auxiliary phage overcomes the steric hindrance effect of p III or p VIII-exogenous fusion proteins and ensures that the recombinant phage can normally infect, assemble and proliferate. The phagemid display system is suitable for displaying antibody molecules and can be used for screening nanobodies with high affinity.
Influenza a virus (Influenza A Virus, IAV) belongs to the genus Orthomyxoviridae (Orthomyxoviridae) and is an enveloped segmented, single-stranded negative-strand RNA virus. The envelope is derived from a host cell membrane, and two kinds of spike glycoprotein, namely Hemagglutinin (HA) and Neuraminidase (NA), are inlaid on the surface of the envelope, and the ratio is about 4:1.HA exists as a trimer on the viral surface, and the receptor binding site (Receptor binding site, RBS) of the trimeric spherical head recognizes and binds to sialoglycosaccharide receptors on the host cell membrane, helping the viral envelope to fuse with the host cell membrane, which is the key to IAV contact and entry into the host cell. NA is mushroom-like tetramer, which can cleave sialic acid and destroy receptor on the surface of host cell membrane, thus promoting release of newly assembled virus particles from host cells in a budding manner, avoiding virus polymerization. IAVs can infect mammals such as birds, humans, pigs, dogs, horses, etc., and are divided into 18 HA subtypes and 11 NA subtypes based on the different antigenicity of HA and NA. Of these, H1N1, H2N2, H3N2 primarily infects humans, while the H5, H7 and H9 subtypes are the most harmful strains to birds. The existing ELISA panning method has the problems that the antigen is not successfully coated or the coated antigen epitope is not fully exposed, and the blocking protein BSA brings steric hindrance, and has universality on enveloped viruses which bud on cell membranes, such as orthomyxoviruses, paramyxoviruses, human immunodeficiency viruses, herpesviruses and the like.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects in the prior art, and provides anti-influenza H9 hemagglutinin nanobodies and an antibody screening method using influenza virus to infect cells.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the invention provides an anti-influenza H9 hemagglutinin nano antibody, which is nano antibody L1-2, nano antibody L1-4 or nano antibody A-5, wherein the amino acid sequence of the nano antibody L1-2 is shown as SEQ ID NO.16, the amino acid sequence of the nano antibody L1-4 is shown as SEQ ID NO.17, and the amino acid sequence of the nano antibody A-5 is shown as SEQ ID NO. 18.
The invention also provides a nucleic acid molecule which codes for the anti-influenza H9 hemagglutinin nanobody.
Further, the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody L1-2 is shown as SEQ ID NO.1, the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody L1-4 is shown as SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody A-5 is shown as SEQ ID NO. 3.
The invention also provides an expression vector comprising the nucleic acid molecule.
The invention also provides a host cell which expresses the anti-influenza H9 hemagglutinin nanobody.
Further, the host cell is Saccharomyces cerevisiae. The host cell is JDY52-Δchs3Saccharomyces cerevisiae.
The invention also provides an antibody screening method for infecting cells by using influenza virus, which is used for screening natural nano antibody libraries after infecting MDCK cells by using avian influenza virus H9N2 (A/chicken/Hebei/L1/2006), and comprises the following steps:
step 1 is cell detoxification: after resuscitating and passaging MDCK cells, adding H9N2 virus liquid for incubation, discarding, and adding cell maintenance liquid for culture;
step 2 is cell forward screening: the recombinant phage adsorbs MDCK cells infected with H9N2, unbound phage is then washed and removed, eluent A containing adsorbed phage and cell debris is collected, the eluent A is proliferated and the titer is determined;
step 3 is cell negative selection: the eluent A is used for adsorbing MDCK cells by bacteriophage, collecting supernatant and eluent to obtain eluent B aiming at influenza virus proteins, proliferating the eluent B and measuring titer;
step 4, repeating the steps 1-3, and screening the nanobody phage display library for the second time and the third time;
step 5 is to determine the recombinant phage enrichment status: detecting phage enrichment states of anti-influenza protein H9-HA in the non-screened phage libraries and the three-wheeled recombinant phage libraries by indirect ELISA;
step 6 is ELISA to check specific nanobody against influenza protein H9-HA: and randomly picking positive monoclonal colonies with different sequences from the titer result of the eluent B, and carrying out ELISA screening of the recombinant protein H9-HA to obtain the nanobody L1-2, the nanobody L1-4 or the nanobody A-5.
Further, the temperature of the infection step in the step 2 is 37 ℃ and the time is 45 minutes; the temperature of the adsorption step in the step 3 is 37 ℃ and the time is 45 minutes.
Influenza virus H9N2 infects MDCK cells, at the end of virus infection, virus proteins HA, NA, M2 and matrix protein M1 are gathered and displayed on the surface of a cell membrane, and a nanobody phage display library carries out forward screening on the infected cells to obtain recombinant phage specific to the cell membrane proteins and the virus proteins; and (3) carrying out negative screening on the surface of uninfected MDCK cells to collect supernatant to obtain recombinant phage with specificity to viral proteins, and removing cell membrane protein specific phage adsorbed by the cells. The method reduces the workload of purifying virus antigen protein, reserves linear epitope and conformational epitope of antigen protein during screening and displaying, overcomes the problems that antigen is not successfully coated or coated antigen epitope is not fully exposed and blocked protein BSA brings steric hindrance, and has universality on enveloped viruses sprouting on cell membranes, such as orthomyxoviruses, paramyxoviruses, human immunodeficiency viruses, herpesviruses and the like.
The invention also provides a construction method of the nanobody phage display library, which comprises the following steps of transfecting a phagemid vector pCANTAB 5E inserted with VHH genes into host bacterium TG1 through a phagemid display system, and obtaining a phage display library of the surface display nanobody through superinfection of helper phage M13KO7, wherein the phage display library comprises the following steps:
step 1 is amplification of VHH gene: extracting RNA from peripheral blood mononuclear cells of alpaca and camel, performing reverse transcription to obtain cDNA, and amplifying VHH genes;
step 2 is the construction of nanobody libraries: double-enzyme cutting plasmid by restriction enzyme, then connecting the VHH gene with double-enzyme cutting carrier, transforming host bacteria by the connection product, culturing and collecting bacteria;
step 3, measuring the library capacity of the nano antibody: performing gradient dilution on the thalli in the step 2, counting single colonies, and calculating library capacity;
step 4 is nanobody phage display library: amplifying and culturing the thalli obtained in the step 2, adding auxiliary phage for adsorption proliferation, and concentrating phage in the supernatant after centrifugation to obtain recombinant phage for displaying nano antibodies;
step 5 is phage titer assay: and (3) after the phage adsorbs the host bacteria, performing single colony counting, and calculating to obtain the recombinant phage titer.
The invention also provides application of the anti-influenza H9 hemagglutinin nano-antibody, and application of the anti-influenza H9 hemagglutinin nano-antibody in preparation of a medicament for treating influenza virus infection; the anti-influenza H9 hemagglutinin nano antibody is applied to preparation of medicaments, reagents, detection plates or kits for detecting influenza virus infection.
The invention codes the 3 strains of anti-influenza protein H9-HA nano antibodies with L1-2, L1-4 and A-5, splices the antibodies with chicken IgY Fc fragment (the sequence is SEQ ID No. 4), inserts the antibody into a yeast secretion type carrier to obtain a POT-alpha factor-VHH-IgY fusion expression carrier, and converts JDY52-Δchs3The saccharomyces cerevisiae chassis cells obtain recombinants, single-chain antibody secretion is verified through Western Blot, antibody titer is detected through indirect ELISA, and in-vitro neutralization activity of the nano-antibodies is detected through virus neutralization experiments.
Compared with the prior art, the invention has the following advantages:
the antibody screening method for infecting cells by using influenza virus disclosed by the invention is based on the phenomenon that virus proteins are gathered on the surface of a cell membrane before the influenza virus budds, and performs positive and negative screening on a natural nano antibody phage display library.
Compared with the conventional ELISA panning method, the antibody screening method for infecting cells by using the influenza virus disclosed by the invention reserves linear epitopes and conformational epitopes of antigen proteins, overcomes the problems of antigen coating failure, insufficient exposure of coated antigen epitopes and steric hindrance brought by blocked proteins in the ELISA panning method, and has universality on enveloped viruses.
The anti-influenza H9 hemagglutinin nano-antibody obtained by the antibody screening method for influenza virus infected cells has a certain neutralizing capacity to H9N2, and provides a potential tool for H9N2 detection and treatment.
Drawings
FIG. 1 is a schematic diagram showing the separation of PBMC from peripheral blood according to example 1 of the present invention;
FIG. 2 shows nested PCR amplification of VHH genes according to example 1 of the present invention: wherein, the A diagram is PCR amplification, and the B diagram is PCR product;
FIG. 3 shows the positive rate of the bacteria liquid PCR identification alpaca (camel) nanobody library in the embodiment 1 of the invention: wherein the A diagram is camel sheep, and the B diagram is alpaca;
FIG. 4 is an ELISA for identifying anti-influenza protein H9-HA phage enrichment as described in example 2 of the present invention;
FIG. 5 is an indirect ELISA for identifying the specificity of 9 recombinant phages for recombinant protein H9-HA according to example 2 of the invention: wherein A is OD 450 The value, B graph is P/N value;
FIG. 6 shows the results of Xba I single enzyme digestion of POT-alpha factor plasmid and chicken IgY Fc gene amplification according to example 3 of the present invention;
FIG. 7 shows the result of PCR amplification of POT-alpha-IgY plasmid bacterial liquid according to example 3 of the present invention;
FIG. 8 shows the result of PCR amplification of POT-alpha-VHH-IgY plasmid bacterial liquid according to example 3 of the present invention;
FIG. 9 is a PCR assay of the VHH-Y recombinant Saccharomyces cerevisiae genome of example 3 of the present invention;
FIG. 10 is a Western Blot analysis of yeast supernatant TCA samples of example 3 of the invention: wherein, the A diagram is L1-3-Y, and the B diagram is L1-3-Y, L1-4-Y and A-5-Y;
FIG. 11 shows the efficacy test of anti-influenza protein H9-HA VHH-IgY single-chain antibodies according to example 4 of the present invention: wherein, A is L1-2-IgY single-chain antibody, B is L1-4-IgY single-chain antibody, and C is A-5-IgY single-chain antibody.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The secretory Saccharomyces cerevisiae strain JDY52-Δchs3The method utilizes homologous recombination to integrate chs3 from Saccharomyces cerevisiae S288C deletion strain library 95401.H2 (self-purchased) to be KanMX4 knockout box, and converts lithium acetate into JDY52 strain to construct and obtain JDY52-Δchs3Strains. The resulting transformants were screened by YPD+G418 and subjected to PCR genotyping and cho3 deleted fold ratio dilution phenotyping. The above methods all belong to the prior art and the target strain can be obtained without doubt.
The secretory Saccharomyces cerevisiae strain JDY52 is disclosed in the article "Guo Y, dong J, zhou T, et al YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae [ J ]. Nucleic Acids Res, 2015,43 (13): e 88".
The present invention will be described in detail with reference to examples.
EXAMPLE 1 construction of a Natural nanobody phage display library
1. Amplification of VHH Gene
1 alpaca and 2 alpaca 10 mL heparin anticoagulants were collected respectively. Adding lymphocyte separation liquid, and centrifuging at room temperature for 2000 r/min and 20 min. After centrifugation, the tube was subjected to liquid stratification, and as a result, the components from top to bottom were plasma, PBMC, lymphocyte separation liquid, and blood cells, as shown in FIG. 1. The PBMCs were extracted with a scissors gun head for total RNA extraction, and cDNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (+gdna wind) reverse transcription.
A first round of PCR amplification was performed using the cDNA obtained by reverse transcription as a template, and the first pair of primers CALL-F (SEQ ID NO. 5) and CALL002 (SEQ ID NO. 6). The 50. Mu.L reaction system was as follows: 2×Hieff Canace Gold PCR Master Mix (YEASEN) 25. Mu.L, cDNA 5. Mu.L, CALL-F2.5. Mu.L, CALL002 2.5. Mu.L, ddH 2 O 15 μL。
Amplification was performed using the following PCR procedure:
98. pre-denaturation at 3 min (denaturation at 98 ℃ C. 10 s. Fwdarw. 70 ℃ C. Annealing 20 s) 15 cycles (1 ℃ C. Reduction per cycle) →72 ℃ C. Extension 15 s. Fwdarw. (denaturation at 98 ℃ C. 10 s. Fwdarw. 55 ℃ C. Annealing 20 s) 15 cycles (1 ℃ C. Reduction per cycle) →72 ℃ C. Extension 15 s. Fwdarw. 72 ℃ C. Extension 5 min.
The PCR amplification yielded two specific bands of sizes 1000 bp and 750 bp, respectively, with the results shown in FIG. 2A. Gel recovery was performed on a strip of about 750 and bp.
The first round of PCR gel recovery product is used as a template, and a second pair of primers VHH-SfiF (SEQ ID NO. 7) and VHH-Not-R (SEQ ID No. 8) amplified VHH gene, 50 μl reaction system as follows: 2 XHieff Canace Gold PCR Master Mix (YEASEN) 25. Mu.L, first round PCR gel recovery product 3. Mu.L, VHH-Sfi-F 2.5 μL，VHH-Not-R 2.5 μL，ddH 2 O 17 μL。
The PCR product size was about 500. 500 bp, and the result is shown in FIG. 2B.
2. Construction of nanobody libraries
Restriction enzyme was used with pCANTAB 5E phagemid vectorSfiI (50 ℃ C.) andNoti (37 ℃) double cleavage, 100. Mu.L cleavage system as follows: pCANTAB 5E phagemid vector 50. Mu.L (5. Mu.g), 10 XQuickCut Buffer 10. Mu.L,NotI 5 μL，ddH 2 O 35 μL。
37. cutting 2 h, and adding 5 mu L of restriction enzymeSfiI, enzyme digestion is carried out at 50 ℃ for 2 h. 100. mu.L of the digested product was subjected to alcohol precipitation at-20℃overnight. 4. Centrifuging at 12000 r/min for 10 min, discarding supernatant, oven drying, and dissolving in 30 μl ddH 2 O, the concentration was measured.
The double digested pCANTAB 5E phagemid vector was ligated to the VHH gene by ClonExpress II One Step Cloning Kit, with 10. Mu.L ligation as follows: 4. Mu.L (90 ng) of double-digested pCANTAB 5E phagemid vector, 1. Mu.L (30 ng) of VHH PCR product, 2. Mu.L of 5 XCE II Buffer, 1. Mu.L of Exnase II and ddH 2 O 2 μL，
The VHH gene of each camel (alpaca) was prepared into an 8-tube 10. Mu.L connection system, and the mixture was connected at 37℃for 30 min after mixing.
Adding 10 μl of the 8-tube connection product of each camel (alpaca) into 8 TG1 pieces, respectively, ice-bathing for 30 min, heat-shock for 1 min at 42 ℃, adding antibiotic-free LB medium, and culturing at 37 ℃ at 180 r/min for 1 h; adding 17 mL of LB-AMP culture medium into the 1 h-branch competent bacteria liquid, and culturing at 37 ℃ at 180 r/min for 6 h; taking 100 mu L of bacterial liquid for library identification, and preserving nano antibody library recombinant plasmid at-20 ℃ by half extracting plasmid of the residual bacterial liquid; the other half of the culture was collected by centrifugation, 0.5 mL of LB medium was resuspended in 0.5 mL of 50% glycerol, and the nanobody library cells were stored at-80 ℃.
3. Determination of nanobody library capacity
Taking the reserved bacterial liquid in the step 2, carrying out 10-fold gradient dilution on 100 mu L of bacterial liquid by using LB-AMP culture medium, and then carrying out 10-fold gradient dilution -4 -10 -6 100 mu L of bacterial liquid is taken and evenly coated on an LB-AMP flat plate, 37. Culturing in an inverted incubator at 10-12 deg.C h; counting the number of single colonies growing on the plates coated with the bacterial solutions with different dilutions, and diluting the bacterial solutions to 10 -5 In this case, 28 single colonies were grown in camel 1 (L1) and 10 single colonies were grown in camel 2 (L2) 6 Individual colonies, alpaca (a) developed 80 individual colonies.
10 single colonies with regular shapes are randomly selected from the flat plate to be cultured in 500 mu L of LB-AMP liquid medium at 37 ℃ and 180 r/min for 5 h; using the universal detection primers M13-R (SEQ ID NO. 9) and VHH-NotR is subjected to bacterial liquid PCR, the positive rate of the nanobody library is identified, the positive rate of the nanobody library of L1 and L2 is 90%, and the positive rate of the nanobody library of A is 100% as shown in figure 3. Calculating the library capacity of the nanobody library:
library size (CFU) =positive rate x colony count x dilution x total volume
The stock capacities of camel 1, camel 2 and alpaca are 6.3X10 respectively 8 CFU、2.39×10 9 CFU and 2.0×10 9 CFU。
4. Rescue natural nano antibody phage display library
(1) 200 mu L of the nano antibody library strains frozen at the temperature of minus 80 ℃ are sucked and added into 30 mL of 2 XYT/2% GLU-AMP culture medium, and the culture is carried out at the temperature of 37 ℃ 180 r/min to the logarithmic phase;
(2) The added titer of 40 mu L is 1.5X10 13 pfu/mL of helper phage M13KO7 (20 MOI), and standing and adsorbing in an incubator at 37 ℃ for 40 min after uniform mixing;
(3) Centrifuging at 25deg.C and 4500 r/min for 20 min, discarding supernatant, re-suspending thallus precipitate with 40 mL 2 XYT/KAN-AMP culture medium, and culturing at 37deg.C and 180 r/min for 3 h;
(4) Centrifuging the cultured bacterial liquid at a temperature of 4 ℃ of 8000 r/min for 30 min, collecting supernatant to a 50 mL centrifuge tube, adding 8 mL of PEG8000/NaCl solution (1/5 supernatant volume), mixing uniformly, and standing on ice for 12-14 h;
(5) Centrifuging at 8500/r/min at 4deg.C for 35 min, discarding supernatant, re-suspending the precipitate with 800 μl PBS buffer, transferring to sterile EP centrifuge tube, incubating at 12000/r/min at 4deg.C for 20 min after shaking table rotation at 4deg.C for 12 h, collecting supernatant, and storing at 4deg.C in dark place.
5. Phage titer assay
Taking 5 mu L of recombinant phage concentrate, and carrying out gradient dilution to 10 by using LB-AMP culture medium -10 -10 -14 The supernatant of each dilution is evenly mixed with equal volume of logarithmic phase TG1 bacterial liquid, and the mixture is coated on an LB-AMP plate after being adsorbed for 30 min in a 37 ℃ incubator, and is inversely cultured at 37 ℃ overnight.
L1 dilution of 10 -12 8 single colonies were grown on LB-AMP plates and the titer of nanobody phage display library was 1.6X10 16 pfu/mL. Dilution of L2, A of 10 -14 The recombinant phage of (2) showed 65 and 25 single colonies on the plate, respectively, and the titer of phage display library was 1.3X10, respectively 19 pfu/mL、5.0×10 18 pfu/mL。
EXAMPLE 2 positive and negative selection of nanobody libraries of influenza virus infected cells
1. Cell detoxification
(1) Cell resuscitation: DMEM culture containing 10% fetal bovine serum is preheated in a water bath kettle at 37 ℃, MDCK cells frozen by liquid nitrogen are quickly thawed by shaking in the water bath kettle at 37 ℃ after being taken out, the thawed cell liquid is added into a cell culture dish containing 10 mL of 10% DMEM culture medium in an ultra clean bench, the cells are evenly spread by cross shaking, and the cells are placed at 37 ℃ and 5% CO 2 Culturing in a cell incubator;
(2) Cell passage: when a monolayer of cells was observed under the microscope to spread on the plate, the medium was discarded in the plate and 1 mL of PBS was added for one rinse in the ultra clean bench, 1 mL trypsin-EDTA digest was added, after waiting 2 min at room temperature, round shrinkage of the cell edges was observed under the microscope, indicating complete digestion, stopping digestion, 1 mL of 10% DMEM medium resuspended cells, 200. Mu.L of cell suspension was added to the plate containing 5 mL of 10% DMEM medium, 600. Mu.L of cell suspension was added to the six well cell culture plate containing 12 mL of 10% DMEM medium, the spread cells were shaken cross-wise and placed in the cell incubator for culturing.
(3) Cell inoculation: when MDCK cells grow to a single layer, the culture solution is discarded in an ultra clean bench, and the culture solution is rinsed 2 to 3 times by PBS, and 500 mu L of DMEM is added into each holeMixing the whole culture medium and 100 μm L H N2 virus solution sterilized by 0.22 μm filter membrane, shaking, standing at 37deg.C, and 5% CO 2 Incubation was performed in a constant temperature incubator for 2 h, the virus solution was discarded, 2 mL of a cell maintenance solution of 1% fetal bovine serum was added, and the incubation was continued in the incubator for 60 h.
2. Cell forward screening
(1) After the cell is inoculated with the virus (H9N 2) 60H, the culture medium is sucked, and PBS is used for washing for three times;
(2) Adsorption: recombinant phage were diluted to 1X 10 with PBS 12 pfu/mL,1 mL/well, 45 min incubation at 37℃and 5 washes with PBS, each with 3 mL of PBS, and standing for 5 min. The negative control was M13KO7;
(3) Eluting: adding 1.5 mL PBS into each hole of a six-hole plate, freezing and thawing once at-80 ℃ in a refrigerator, collecting eluent A containing adsorbed phage and cell fragments, and respectively measuring the titer of the eluent A and the titer of a negative control eluent;
(4) Proliferation eluent a: taking 2 mL phage eluent A to adsorb 6 mL log phase TG1 bacterial liquid, and multiplying phage of eluent A.
3. Negative cell selection
(1) Removal of cell surface protein specific phages: eluent A phage was diluted to 1X 10 in PBS after proliferation 12 pfu/mL,1 mL/well are added into a six-hole plate for culturing non-virus MDCK cells, incubation is carried out at 37 ℃ for 45 min, supernatant is collected, PBS is washed 3 times, 1 mL is added each time and kept stand for 5 min, eluent is collected, eluent B (supernatant and three eluents) aiming at influenza virus proteins is obtained, and the titer of recombinant phage of the eluent B is measured;
(2) Proliferation eluent B: adsorbing 6 mL log phase TG1 bacterial liquid by using 3 mL phage eluent B, and proliferating phage of the eluent B;
(3) Repeating the steps (1) - (3), and carrying out second and third screening on the nanobody phage display library.
4. Determination of recombinant phage enrichment status
(1) Tanning: 200 mu L/hole sample adding of 96-hole ELISA plate with 0.2% tannic acid, tanning for 2-3 h in a incubator at 37 ℃ and ddH 2 O is washed for 2 times;
(2) Antigen coating: the CB coating liquid dilutes recombinant antigen protein H9-HA to 4 mug/mL, 100 mug L/Kong Baojia sample, and the amino acid sequence of the recombinant protein H9-HA is SEQ ID NO.10.CB coating solution served as negative control. 4. Coating 12 h overnight. PBST is washed 3 times;
(3) Closing: 3% BSA 100 [ mu ] L/well was loaded, blocked at 37℃for 1 h, and PBST was washed 3 times for 3 min each; titer of 3% BSA diluted non-selected phage library and three rounds of recombinant phage library to 5X 10 11 pfu/mL, room temperature spin-closure 1 h;
(4) Incubating phage: and (5) loading 100 mu L/hole of the closed recombinant phage concentrate. 37. Incubate at C for 30 min. PBST is washed for 3 times, each time for 3 min;
(5) Enzyme conjugate addition: after dilution of Anti-M13 Anti-body (HRP) to 0.1-0.4. Mu.g/mL, 100. Mu.L/well was loaded. 37. Incubate at C for 30 min. PBST is washed for 3 times, each time for 3 min;
(6) Color development: adding 100 mu L of TMB single-component color development liquid into each hole, and carrying out light-proof reaction for 12-18 min in a 37 ℃ incubator;
(7) And (3) terminating: after adding 50. Mu.L of stop solution per well, the absorbance at 450 nm was measured by a microplate reader. The results are shown in FIG. 4, OD of three rounds of specific recombinant phages 450 The value appears to rise regularly. It was demonstrated that specific phages against influenza H9-HA were enriched in three rounds of cell screening.
5. ELISA check anti-influenza protein H9-HA specific nanobody
(1) Phage for obtaining specific nanobodies:
10 monoclonal colonies were randomly picked from the third round of titer plate of cell selection eluate B and cultured in 500. Mu.L LB-AMP medium at 37℃180 r/min for 5 h. Colony PCR was performed as in example 1, step 3. And (3) sending bacterial liquid of positive clones to a Jin Weizhi company for sequencing, and selecting nano antibodies with different amino acid sequences according to a sequencing result, wherein the numbers of the 9 nano antibodies are L1-2, L1-3, L1-4, L1-8, L2-2, L2-5, A-3, A-5 and A-6 respectively. A single specific phage concentrate was obtained as in example 1, step 4.
2. ELISA check of specific phages:
the recombinant protein H9-HA was coated, and ELISA screening was performed according to the steps (1) to (7) in step 4, and the results are shown in FIG. 5. The P/N value of L1-2, L1-4 and A-5 is more than 2.0, and can be combined with recombinant H9-HA; the P/N values of L1-8, L2-5, A-3 and A-6 are greater than 1.5, and the possibility of binding to recombinant H9-HA protein exists; the P/N values of L1-3 and L2-2 are less than 1.5, indicating that it is not specific for recombinant H9-HA, possibly that recombinant H9-HA does not contain this antibody recognition epitope, or is a nanobody potentially recognizing other influenza virus proteins (e.g., NA).
EXAMPLE 3 construction of Yeast recombinant secreting anti-influenza H9-HA Single chain antibody
1. Chicken IgY Fc gene amplification
The chicken cDNA obtained by reverse transcription is used as a template, and IgY Fc-F (SEQ ID NO. 11) and IgY Fc-R (SEQ ID NO. 12) are used as primers for amplification. The 50. Mu.L reaction system was as follows: 2×Hieff Canace Gold PCR Master Mix (YEASEN) 25. Mu.L, cDNA 1. Mu.L, igY Fc-F2.5. Mu.L, igY Fc-R2.5. Mu.L ddH 2 O 19 μL。
The PCR procedure was performed as in example 1, step 1, with a PCR product size of 1000 bp, and the results are shown in FIG. 6.
2. Construction of VHH-IgY Fc fusion expression vector
XbaThe results of the single cleavage of the POT vector are shown in FIG. 6, and the chicken IgY Fc gene is seamlessly cloned and connected with the single cleavage of the POT vector to transform TOP10 competent cells. After coating LB-AMP plates, the next day, plates were picked up 10 single colonies and inoculated into 500. Mu.L LB-AMP medium, incubated at 37℃180R/min for 5 h, bacterial liquid PCR was performed using primers α -factor-F (SEQ ID NO.13: GGGGATTTCGATGTTGCT) and IgY Fc-R, bacterial liquid PCR verification was performed, and as shown in FIG. 7, the bacterial liquid PCR target band of chicken was 1100 bp or so, and POT- α -IgY plasmids were extracted.
XbaThe primers alpha-VHH-F (SEQ ID NO. 14) and IgY-VHH-R (SEQ ID NO. 15) were amplified to obtain VHH genes with homology arms, which were ligated with POT-alpha-IgY vectors to transform TOP10 competent cells using 3 specific VHHs (L1-2, L1-4, A-5) and non-recombinant H9-HA specific L1-3 screened in step 5 of example 2 as templates. Bacterial liquid PCR was performed using primers α -factor-F and IgY-VHH-R, and the results are shown in FIG. 8As shown, the bacterial liquid PCR target band of chicken is about 500 bp, and POT-alpha-L1-2-IgY, POT-alpha-L1-4-IgY, POT-alpha-A-5-IgY and POT-alpha-L1-3-IgY plasmids are extracted, and the plasmids are disclosed in "Han Zhang, zexing Li, et al, recombinant hemagglutinin displaying on yeast reshapes congenital lymphocyte subsets to prompt optimized systemic immune protection against avian in fl uenza in microbiology 2023.14 DOI: 10.3389/fmicb.2023.1153922".
3. Construction of recombinant Saccharomyces cerevisiae
(1) Construction of secretory Saccharomyces cerevisiae transcription unit
BsaCleavage of POT-alpha-VHH-IgY and negative control POT-alpha-L1-3-IgY 20. Mu.L of the cleavage system was prepared as follows. After mixing, enzyme cutting is carried out at 37 ℃ for 3 h. POT recombinant plasmid 8. Mu.L (500 ng), cutSmart (New England Biolabs). Mu.L,BsaI（New England Biolabs）1 μL，ddH 2 O 9 μL，
by usingBsmBI cleavage three pairs of homology arm plasmids (URR 1/URR2, TU/TD, CIP/HST) and selectable marker plasmid (PMV-TRP), one pair of homology arms/TRP formulated 1 20. Mu.L cleavage system. After mixing, enzyme cutting is carried out at 55 ℃ for 3 h.
PMV-URR1/TU/CIP 3 μL（500 ng），PMV-URR2/TD/HST 3 μL（500 ng），PMV-TRP 2 μL（500 ng），NEB Buffer 3.1 2 μL，BsmBI（New England Biolabs）1 μL，ddH 2 O 9 μL，
After completion of cleavage, 2. Mu.L of Lcom/Rsm (10. Mu.M) was added to the homology arm/TRP cleavage system, and 2. Mu.L of Lcom/Rcom (10. Mu.M) was added to the transcription unit cleavage system. Both systems were run in a PCR instrument with the following procedure:
83 ℃ 6 min，80 ℃ 3 min，75 ℃ 1.5 min，70 ℃ 1.5 min，65 ℃ 1.5 min，60 ℃ 1.5 min，55 ℃ 1.5 min，50 ℃ 1.5 min。
the transcription unit and the cleavage system of the homology arm were linked by T4 ligase, and 50. Mu.L of the ligation system was as follows. After mixing, the mixture was allowed to stand at 16℃overnight. Homology arm/TRP cleavage product 24. Mu.L, transcription unit cleavage product 24. Mu.L, 10×T4add on buffer 5. Mu.L, T4 DNA library (Thermo) 1. Mu.L.
(2) Yeast transformation
From the secretory Saccharomyces cerevisiae strain JDY52-Δchs3Single colonies were picked up on plates and inoculated into 3 mL YPD medium at 30℃and 200 r/min overnight; the following day, 200 mu L of the overnight culture bacterial liquid is taken according to the proportion of 1:50 and inoculated into 10 mL of YPD culture medium, and the culture is carried out at 30 ℃ and 200 r/min until the OD value reaches 0.6;5000 Centrifuging at r/min for 1 min, collecting thallus into 1 EP tube, adding 1 mL ddH 2 O re-suspending the thalli, centrifuging again, and discarding the supernatant; adding 100 mu L of 0.1-M LiAc into the precipitate, shaking and uniformly mixing, centrifuging at 12000 r/min for 20 s, and discarding the supernatant; adding 50 mu L of 0.1M LiAc again, shaking, mixing uniformly, centrifuging at 12000 r/min for 20 s, and discarding the supernatant; sequentially adding 240 mu L of 50% PEG4000, 36 mu L of 1M LiAc, 20 mu L of 10 mg/mL ssDNA (ice bath after 5 min of sample boiling in boiling water) and 50 mu L of the connection product into an EP tube, and carrying out vortex shaking for 1 min until the connection product is completely mixed uniformly, and culturing at 30 ℃ for 30 min at 200 r/min; 42. after heat shock for 30 min at the temperature of 6000 r/min, centrifuging for 30 s to collect thalli, adding 1 XYPD medium of mL, culturing at 30 ℃ at 200 r/min for 2 h; and then centrifuging at 5000 r/min for 1 min to collect thalli, leaving 100 mu L of resuspended thalli, coating on an SD-TRP plate, and placing in a 30 ℃ incubator for 2-3 days. Single colonies on the plates were picked and inoculated into 500. Mu.L of YPD liquid medium, cultured overnight at 30℃and 200. 200 r/min, and the bacterial solutions were used for genotyping.
(3) Testing of Yeast genotypes
Taking 100 mu L of yeast liquid cultured overnight, centrifuging for 1 min at 12000 r/min, discarding the supernatant, and adding 100 mu L of 200 mu M Solution I (formula of 200 mu L of 1M LiAc, 100 mu L of 10% SDS, 700 mu L of ddH 2 O); 90. heat shock is carried out for 10 min at the temperature, then 300 mu L of absolute ethyl alcohol is added, the mixture is evenly mixed upside down, and the mixture is stood for 5 min;12000 Centrifuging for 3 min at r/min to precipitate DNA, and discarding supernatant; adding 400 mu L of 75% ethanol, standing for 1 min, centrifuging for 1 min at 12000 r/min, and discarding the supernatant; after airing, 50 mu L ddH 2 O is subjected to resuspension precipitation, 2 mu L is taken as a template for PCR verification, the PCR detection primer is alpha factor-F/IgY Fc-R, the result is shown in figure 9, and the target band is about 1500 bp.
4. Verification of expression conditions of recombinant Saccharomyces cerevisiae
(1) Yeast culture expression
A single colony of recombinant yeast positive in genotype verification was inoculated into 3 mL of YPD medium and cultured at 30℃and 200 r/min for 72 h.
(2) TCA sample preparation
Trichloroacetic acid (TCA) was dissolved in a water bath in advance, 1.8. 1.8 mL yeast supernatant was collected from the centrifuged solution, 1/9 volume of TCA (200. Mu.L) was added, and the mixture was mixed upside down to give an ice bath 1 h. 4. Centrifuging at 12000/r/min for 10 min, discarding supernatant, air drying, and making precipitate brown. Adding 30 μl of 0.5M NaOH solution to dissolve the precipitate, adding 7.5 μl of 5×loading Buffer, boiling with boiling water for 10 min, centrifuging at 12000 r/min for 1 min, and sucking supernatant.
(3) Western Blot detection of recombinant Saccharomyces cerevisiae expression
SDS-PAGE electrophoresis: absorbing 10 mu L of protein sample supernatant, adding sample, and after 80V constant pressure running concentrated gel for 30 min, raising the sample to 130V constant pressure running separated gel for 60 min;
B. transferring the protein adhesive tape to a PVDF film activated by methanol by a wet transfer method, and transferring the film to a 300 mA constant flow film for 100 min; placing PVDF membrane in 5% skimmed milk, and sealing with shaker 70 r/min at room temperature for 1 h; immersing a PVDF membrane in a mouse anti-His monoclonal antibody (1:5000 dilution), and incubating the primary antibody at 4 ℃ overnight; recovering primary antibody, washing with 1 XTBE, repeating for 3 times, shaking table 90 r/min for 10 min each time; the membrane was placed in HRP-goat anti-mouse IgG antibody (1:5000 dilution), and secondary antibody 1 h was incubated on a shaker at room temperature of 70 r/min; recovering secondary antibody, washing film with 1 XTBE, repeating 3 times, shaking table 90 r/min each time for 10 min; and (3) dropwise adding a substrate color developing solution to the front surface of the film, and performing exposure color development by a chemiluminescent imager.
As shown in FIG. 10, the VHH-IgY Fc single-chain antibody was successfully secreted into the yeast supernatant, and the molecular weight was about 58kDa, and the size was correct.
Example 4 detection of anti-influenza protein H9-HA Single chain antibody
1. ELISA detection of anti-influenza H9-HA VHH-IgY single-chain antibody titer
(1) Concentrating the supernatant of the recombinant yeast fermentation broth: 10 mL is cultured to be 1 mL from recombinant yeast supernatant of L1-2-Y, L1-4-Y and A-5-Y of 72 h, and the yeast fermentation broth is replaced by PBS buffer solution to obtain 1 mL isotonic solution containing secreted protein; the negative control selected recombinant yeast L1-3-Y that was not specific for recombinant H9-HA.
(2) ELISA detection:
after tanning of the 96-hole ELISA plate, the CB coating liquid dilutes and recombines H9-HA to 5 mug/mL, and 100 mug L/Kong Baojia samples are taken. 4. Coating for 12 h overnight at the temperature of between three times, and washing with PBST for 3 times; 3% BSA 100 [ mu ] L/hole is sampled, the sample is blocked for 1 h at 37 ℃, PBST is washed 3 times, and each time is 2-3 min; and (3) adding 200 mu L/hole of the concentrated solution of the detected antibody supernatant diluted by 2 times, and incubating for 30 min at the temperature of 37 ℃. PBST is washed for 3 times, each time for 3 min; adding sheep anti-chicken IgG-HRP diluted according to the ratio of 1:5000, incubating for 30 min at 37 ℃, and washing with PBST for 3 times each for 3 min; the absorbance at 450 nm was measured by a post-development microplate reader and the results are shown in FIG. 11.
ELISA titers of the L1-2-IgY, L1-4-IgY and A-5-IgY single-chain antibodies on H9N2-HA were 1:64,1:32 and 1:32, respectively.
2. Virus neutralization assay
(1) Virus TCID 50 And (3) measuring: after 10-fold serial dilutions of the virus solution, the virus solution was added to 96-well cell culture plates grown to a monolayer of MDCK cells, each dilution was inoculated with 8-well cells, 100. Mu.L per well, and TCID was measured by the Reed-Muench method 50 。
(2) The supernatant of the 15 mL recombinant yeast broth was concentrated to 1 mL and replaced with PBS buffer and sterilized with a 0.45 μm filter.
(3) mu.L of the concentrated yeast fermentation supernatant was mixed with 500. Mu.L of the cell maintenance solution, and diluted 2-fold with the cell maintenance solution. To 500. Mu.L of the maintenance solution at each dilution was added 500. Mu.L of 400 TCID 50 Is incubated at 37℃for 1H. 100. Mu.L of the mixture was added to a 96-well cell culture plate, and 8-well cells were inoculated at each dilution. Marking, placing 96-well cell culture plate in 5% CO 2 After 48 and h culture in a 37 ℃ incubator, cytopathic effect (Cytopathic effect, CPE) is observed, and half protection amount PD is calculated by using a Reed-Muench method 50 。
The virus neutralization experimental result of the recombinant yeast supernatant shows that the PD of the single-chain antibody of the L1-2-IgY, the L1-4-IgY and the A-5-IgY 50 1:45,1:23,1:33.The experimental results show that L1-2, L1-4 and A-5 are nanobodies which recognize H9-HA and have neutralizing capacity to influenza virus H9N 2.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An anti-influenza H9 hemagglutinin nanobody, characterized in that: the anti-influenza H9 hemagglutinin nano antibody is a nano antibody L1-2, a nano antibody L1-4 or a nano antibody A-5, the amino acid sequence of the nano antibody L1-2 is shown as SEQ ID NO.16, the amino acid sequence of the nano antibody L1-4 is shown as SEQ ID NO.17, and the amino acid sequence of the nano antibody A-5 is shown as SEQ ID NO. 18.
2. A nucleic acid molecule characterized in that: the nucleic acid molecule encodes the anti-influenza H9 hemagglutinin nanobody of claim 1.
3. The nucleic acid molecule of claim 2, wherein: the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody L1-2 is shown as SEQ ID NO.1, the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody L1-4 is shown as SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule for encoding the nano antibody A-5 is shown as SEQ ID NO. 3.
4. An expression vector, characterized in that: the expression vector comprising the nucleic acid molecule of claim 2 or 3.
5. A host cell, characterized in that: the host cell expresses the anti-influenza H9 hemagglutinin nanobody of claim 1.
6. The host cell of claim 5, wherein: the host cell is Saccharomyces cerevisiae.
7. An antibody screening method for infecting cells with influenza virus, characterized in that: the method comprises the following steps:
step 1 is cell detoxification: after resuscitating and passaging MDCK cells, adding H9N2 virus liquid for incubation, discarding, and adding cell maintenance liquid for culture;
step 2 is cell forward screening: the recombinant phage adsorbs MDCK cells infected with H9N2, unbound phage is then washed and removed, eluent A containing adsorbed phage and cell debris is collected, the eluent A is proliferated and the titer is determined;
step 3 is cell negative selection: the eluent A is used for adsorbing MDCK cells by bacteriophage, collecting supernatant and eluent to obtain eluent B aiming at influenza virus proteins, proliferating the eluent B and measuring titer;
step 4, repeating the steps 1-3, and performing second and third screening on the nanobody phage display library according to claim 6;
step 5 is to determine the recombinant phage enrichment status: detecting phage enrichment states of anti-influenza protein H9-HA in the non-screened phage libraries and the three-wheeled recombinant phage libraries by indirect ELISA;
step 6 is ELISA to check specific nanobody against influenza protein H9-HA: and randomly picking positive monoclonal colonies with different sequences from the titer result of the eluent B, and performing ELISA screening of the recombinant protein H9-HA to obtain the nanobody L1-2, the nanobody L1-4 or the nanobody A-5 in claim 1.
8. The method for screening an antibody against influenza virus-infected cells according to claim 7, wherein: the temperature of the infection step in the step 2 is 37 ℃ and the time is 45 minutes; the temperature of the adsorption step in the step 3 is 37 ℃ and the time is 45 minutes.
9. A construction method of a nanobody phage display library is characterized by comprising the following steps: the method comprises the following steps:
step 1 is amplification of VHH gene: extracting RNA from peripheral blood mononuclear cells of alpaca and camel, performing reverse transcription to obtain cDNA, and amplifying VHH genes;
step 2 is the construction of nanobody libraries: double-enzyme cutting plasmid by restriction enzyme, then connecting the VHH gene with double-enzyme cutting carrier, transforming host bacteria by the connection product, culturing and collecting bacteria;
step 3, measuring the library capacity of the nano antibody: performing gradient dilution on the thalli in the step 2, counting single colonies, and calculating library capacity;
step 4 is nanobody phage display library: amplifying and culturing the thalli obtained in the step 2, adding auxiliary phage for adsorption proliferation, and concentrating phage in the supernatant after centrifugation to obtain recombinant phage for displaying nano antibodies;
step 5 is phage titer assay: and (3) after the phage adsorbs the host bacteria, performing single colony counting, and calculating to obtain the recombinant phage titer.
10. The use of the anti-influenza H9 hemagglutinin nanobody of claim 1 for the preparation of a medicament for the treatment of influenza virus infection or for the preparation of a medicament, reagent, assay plate or kit for the detection of influenza virus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311167339.6A CN116903733B (en) | 2023-09-12 | 2023-09-12 | Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311167339.6A CN116903733B (en) | 2023-09-12 | 2023-09-12 | Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116903733A true CN116903733A (en) | 2023-10-20 |
| CN116903733B CN116903733B (en) | 2023-12-15 |
Family
ID=88353477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311167339.6A Active CN116903733B (en) | 2023-09-12 | 2023-09-12 | Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116903733B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872351A (en) * | 2019-09-06 | 2020-03-10 | 广西科技大学 | Nano antibody GN1 specifically bound with GPC3 protein and preparation method and application thereof |
| CN111320694A (en) * | 2020-02-15 | 2020-06-23 | 广西科技大学 | Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof |
| CN113735968A (en) * | 2020-12-08 | 2021-12-03 | 山东畜牧兽医职业学院 | Method for measuring titer of swine transmissible gastroenteritis virus N protein antibody |
-
2023
- 2023-09-12 CN CN202311167339.6A patent/CN116903733B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872351A (en) * | 2019-09-06 | 2020-03-10 | 广西科技大学 | Nano antibody GN1 specifically bound with GPC3 protein and preparation method and application thereof |
| CN111320694A (en) * | 2020-02-15 | 2020-06-23 | 广西科技大学 | Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof |
| CN113735968A (en) * | 2020-12-08 | 2021-12-03 | 山东畜牧兽医职业学院 | Method for measuring titer of swine transmissible gastroenteritis virus N protein antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116903733B (en) | 2023-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111995675A (en) | Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof | |
| CN111303255B (en) | COVID-19-S-RBD virus-like particle, vaccine and preparation method thereof | |
| CN111848786B (en) | Monoclonal antibody, preparation method and application thereof | |
| CN111171146B (en) | Nano antibody for resisting H9N2 subtype avian influenza virus, preparation method and application | |
| CN110776564B (en) | Two-strain anti-newcastle disease virus nano antibody and expression preparation method and application thereof | |
| CN111057145A (en) | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof | |
| CN113150083A (en) | Recombinant avian influenza subunit vaccine and preparation method thereof | |
| CN106188283B (en) | Nano antibody of avian influenza A H7N2 and application thereof | |
| CN110577594B (en) | Staphylococcus aureus enterotoxin A nano antibody A21, application and kit | |
| CN114957454A (en) | Nano antibody and fusion protein for resisting CSFV E2 protein, and preparation method and application thereof | |
| CN116903733B (en) | Anti-influenza H9 hemagglutinin nanobody and antibody screening method for cells infected by influenza virus | |
| CN110568189B (en) | Dog adenovirus type 1 antibody ELISA detection kit and application thereof | |
| CN116589569B (en) | Nanometer antibody of H7N9 subtype influenza virus HA protein and construction method and application of phage display library thereof | |
| CN116462754A (en) | Monoclonal antibody for identifying N protein of canine distemper virus, detection reagent and application | |
| CN113717284B (en) | Hepatitis A virus specific nano antibody and application thereof | |
| CN110452885B (en) | Hybridoma cell 4F6 strain secreting monoclonal antibody against newcastle disease virus NP protein | |
| CN111825763B (en) | Influenza virus HA protein stem specific monoclonal antibody and preparation method and application thereof | |
| CN110161246B (en) | Indirect competitive ELISA method and kit for distinguishing Newcastle disease virus-like particle vaccine immune serum and wild virus infection serum | |
| CN111454171B (en) | Paraquat hapten PH-A, artificial antigen and antibody as well as preparation methods and applications thereof | |
| CN114113634A (en) | ELISA detection kit for detecting African swine fever virus antibody and application of protein L | |
| CN113583119A (en) | Anti-staphylococcus aureus nanobody Nb56, application and kit | |
| CN117164705B (en) | Nanometer antibody of H5 subtype avian influenza virus hemagglutinin protein | |
| CN112940081B (en) | Polypeptide molecule capable of specifically binding novel coronavirus nucleocapsid protein and preparation method thereof | |
| CN111499734B (en) | Single-chain antibody for resisting duck circovirus and preparation method and application thereof | |
| CN110669129B (en) | Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |