CN106190986B - The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody - Google Patents
The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody Download PDFInfo
- Publication number
- CN106190986B CN106190986B CN201610533234.1A CN201610533234A CN106190986B CN 106190986 B CN106190986 B CN 106190986B CN 201610533234 A CN201610533234 A CN 201610533234A CN 106190986 B CN106190986 B CN 106190986B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- albumen
- hybridoma cell
- btv4
- cell strain
- 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.)
- Active
Links
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 29
- 241000120506 Bluetongue virus Species 0.000 title claims abstract description 12
- 210000002966 serum Anatomy 0.000 title claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 230000028327 secretion Effects 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 239000013598 vector Substances 0.000 abstract description 6
- 238000010276 construction Methods 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 2
- 102000036639 antigens Human genes 0.000 abstract description 2
- 108091007433 antigens Proteins 0.000 abstract description 2
- 230000002163 immunogen Effects 0.000 abstract description 2
- 238000002405 diagnostic procedure Methods 0.000 abstract 1
- 230000029087 digestion Effects 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 241001465754 Metazoa Species 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 208000003836 bluetongue Diseases 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000009465 prokaryotic expression Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 101150027802 L2 gene Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 241000702259 Orbivirus Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 241000702247 Reoviridae Species 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 210000003000 inclusion body Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 206010003645 Atopy Diseases 0.000 description 1
- 241000347416 Bluetongue virus 4 Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001416149 Ovis ammon Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- -1 benzene Diamines Chemical class 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000005178 buccal mucosa Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of monoclonal antibodies of 4 type VP2 albumen of resistant to bluetongue virus serum, secrete the hybridoma cell strain and purposes of the monoclonal antibody.The VP2-4B albumen that the present invention is expressed using pET-28a as vector construction is as immunogen immune mouse, the VP2-4B albumen expressed using pMAL-c5x as vector construction is as antigen, screen positive hybridoma cell, it finally screens to obtain one plant of hybridoma cell strain for capableing of the anti-BTV4-VP2 protein monoclonal antibody of stably excreting, experiments have shown that, with BTV4-VP2 albumen specific reaction can occur for monoclonal antibody BTV4-VP2-1B6 secreted by the hybridoma cell strain, and not react with other serotypes VP2 albumen.Monoclonal antibody BTV4-VP2-1B6 of the invention is to establish BTV4 and the serology differential diagnostic method of other serotypes is laid a good foundation.
Description
Technical field
The present invention relates to the monoclonal antibody more particularly to one plant of secretion of a strain of hybridoma strain and its secretion are anti-
The hybridoma cell strain of BTV4VP2 protein monoclonal antibody and its secreted monoclonal antibody；The invention further relates to above-mentioned miscellaneous
The application of tumor cell strain, monoclonal antibody in preparation diagnosis or prevention BTV4 infection detection is handed over, the prevention and treatment neck of blue tongue disease is belonged to
Domain.
Background technique
Blue tongue disease (Bluetongue, BT) is by the blue tongue virus of Reoviridae Orbivirus
Instead when animal insect-borne infectious disease caused by (Bluetonguevirus, BTV).Blue tongue disease is a kind of acute untouchable infectious disease,
It is mainly characterized by with fever, oligoleukocythemia, buccal mucosa and gastrointestinal tract mucous serious catarrhal inflammation.BTV can infect mostly
Number is domestic and wild instead to work as animal, because the neurological susceptibility different manifestations of animal go out different clinical symptoms.BTV main infection is continuous
Sheep, ox etc. is domestic and wild instead when class animal, the death rate 5%-30% anti-after zoogenetic infection BTV are differed, and susceptible sheep can
Up to 80%.BT is classified as legal notification disease by the influence due to BT to world economy, World Organization for Animal Health (OIE),
China delimited as a kind of animal epidemic.On the one hand the loss of related economic caused by BT passes through the production of directly reduction animal
Ability and the death rate for increasing animal, it is often more important that due to the flowing of control animal, the outlet of control ox sperm and implement this
Expense needed for a little control measure and indirectly caused by animal trade lose.
Blue tongue virus (Bluetongue virus, BTV) belongs to Reoviridae (Reoviridae) Orbivirus
Belong to (Orbivirus) blue tongue virus subgroup (Bluetongue virus subgroup).BTV is double-stranded RNA (dsRNA) disease
Poison, genome are made of 10 linear dsRNA segments, and the total genome of BTV encodes 11 hatching eggs there are about 19,200bp composition altogether
It is white, including 7 kinds of structural proteins (VP1-VP7) and 4 kinds of non-structural proteins (NS1, NS2, NS3/NS3a, NS4).
The internal layer capsid of BTV is mainly made of VP3 albumen, is encoded by L3, around three secondary albumen VP1, VP4, VP6
The non-central of virus is constituted with 10 genomic fragments；VP7 albumen constitutes the intermediate capsid of BTV, is encoded by S7, the albumen and Asia
Core component and viral internal other compositions are assembled into the core of virus jointly；The outer layer clothing of VP2 and VP5 albumen composition virus
Shell is encoded by L2 and M5 respectively.And four kinds of non-structural proteins mainly appear in infected cell.
BTV serotype is numerous, has now been found that 24 serotypes, and as the new serotype of climate change continuously emerges.
However due between each serotype Cross immunogenicity it is poor so that vaccine immunity is intricate cannot to play good protection
Effect, up to the present there has been no the vaccines that effectively can be used for preventing and treating blue tongue disease.Early stage Accurate Diagnosis, early prevention are anti-
The only most effectual way of this disease outburst.Therefore it is necessary to reinforce the research of the related work of China BT, necessary technology storage is carried out
It is standby.
Summary of the invention
One of the object of the invention be to provide one plant of 4 type of secretion resistant to bluetongue virus serum (Bluetonguevirus 4,
BTV4) the hybridoma cell strain of VP2 protein monoclonal antibody；
The second object of the present invention is to providing a kind of monoclonal antibody as secreted by above-mentioned hybridoma cell strain, the monoclonal
With BTV 4-VP2 albumen specific reaction can occur for antibody；
The three of the object of the invention are to provide said monoclonal antibody and are preparing the purposes detected in BTV4 type.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The VP2-4B albumen that the present invention is expressed using pET-28a as vector construction as immunogen immune mouse, take it is immune after
Mouse spleen lymphocyte and SP2/0 cell fusion.In addition, the VP2-4B that the present invention is also expressed using pMAL-c5x as vector construction
Albumen screens to obtain the anti-BTV4-VP2 protein monoclonal antibody of one plant of stably excreting through indirect ELISA method as envelope antigen
Hybridoma cell strain, be named as hybridoma cell strain 1B6, classification naming is hybridoma cell strain 1B6；It is deposited in Chinese Typical Representative
Culture collection, address is in Wuhan University, microbial preservation number are as follows: CCTCC No.C2016107, preservation time is
On July 1st, 2016.
The present invention also provides a kind of monoclonal antibodies as secreted by above-mentioned hybridoma cell strain, are named as BTV4-
VP2-1B6, identified monoclonal antibody BTV4-VP2-1B6 are IgG1 hypotype, and light chain is Kappa chain.Western blot inspection
It surveys the result shows that with BTV4-VP2 albumen specific reaction can occur for BTV4-VP2-1B6, and not react with SP2/0 cell.
Further, it is being prepared the invention also provides the hybridoma cell strain and by its monoclonal sports secreted
Detect the purposes in BTV4 virus agent.
In conclusion the present invention prepares and identifies the monoclonal antibody that a species specificity is directed to BTV4-VP2 albumen,
Monoclonal antibody of the invention is to establish the research of BTV4 type specificity immunological detection method to lay a good foundation.
Detailed description of the invention
Fig. 1 is the qualification result of pET-28a-4A, pET-28a-4B and pET-28a-4C；
1.Trans2K PlusⅡDNA Marker；2.pET-28a-4A plasmid；3.pET-28a-4A through the single enzyme of BamH I
It cuts；4.pET-28a-4A is through I double digestion of BamH I and Xho；5.pET-28a-4B plasmid；6.pET-28a-4B is single through BamH I
Digestion；7.pET-28a-4B is through I double digestion of BamH I and Xho；8.pET-28a-4C plasmid；9.pET-28a-4C is through BamH I
Single endonuclease digestion；10.pET-28a-4C through BamH I and XhoI double digestion；
Fig. 2 is the qualification result of pMAL-c5x-4A, pMAL-c5x-4B and pMAL-c5x-4C；
1.Trans2K PlusⅡDNA Marker；2.pMAL-c5x-4A plasmid；3.pMAL-c5x-4A is through the single enzyme of Sal I
It cuts；4.pMAL-c5x-4A through I double digestion of Sal I and Not；5.pMAL-c5x-4B plasmid；6.pMAL-c5x-4B is single through Sal I
Digestion；7.pMAL-c5x-4B through I double digestion of Sal I and EcoR；8.pMAL-c5x-4C plasmid；9.pMAL-c5x-4C is through Sal
I single endonuclease digestion；10.pMAL-c5x-4C is through I double digestion of Sal I and EcoR；
Fig. 3 is the SDS-PAGE analysis result for recombinating His-4B albumen；
Before 1.pET-28a-4B/BL21 induction；After 2.pET-28a-4B/BL21 induction；3. protein molecular weight standard；
Fig. 4 is the SDS-PAGE analysis result for recombinating MBP-4B albumen；
1. protein molecular weight standard；Before 2.pMAL-c5x-4B/BL21 induction；After 3.pMAL-c5x-4B/BL21 induction；
Fig. 5 is the Western blot analysis result of His-4B purifying protein and His label monoclonal antibody；
1.His-4B purifying protein；2 protein molecular weight standards；
Fig. 6 is monoclonal antibody 1B6 and BTV4-VP2-A, BTV4-VP2-B and BTV4-VP2-C of the invention
Western blot analyzes result.
1.BTV4-VP2-A；2.BTV4-VP2-B；3:BTV4-VP2-C；4. protein molecular weight standard.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Main experimental materials involved in the embodiment of the present invention and source:
1, main agents and drug
Various restriction enzymes, T4 ligase, reverse transcriptase, Taq enzyme, DNA marker are that precious bioengineering is (big
Even) Co., Ltd's product；Small amount plasmid extraction agent box purchases AXYGE company；Glue recycling small volume of reagent box has purchased from middle Ke Ruitai
Limit company；Horseradish peroxidase (HRP) marks sheep anti-mouse igg to be purchased from Bioisystech Co., Ltd, Beijing Zhong Shan Golden Bridge；Adjacent benzene
Diamines (OPD) is purchased from SIGMA company；Fetal calf serum is purchased from GIBCO company；Superfine fetal calf serum is purchased from Tianjin Hao sun company；
1640 basal mediums, dimethyl sulfoxide (DMSO), trypsase, PEG3350, Freund's complete adjuvant and Freund are not exclusively helped
Agent, HAT (50 ×) culture medium, HT (l00 ×) culture medium, diaminobenzidine (DAB), o-phenylenediamine (OPD), Subclass of antibody mirror
Kit is determined purchased from SIGMA company.
2, experimental animal
4-6 week old BALB/C mice is provided by the long-living Bioisystech Co., Ltd in Liaoning.
The prokaryotic expression of 1 4BTV-VP2-A, 4BTV-VP2-B and 4BTV-VP2-C albumen of embodiment and purifying
1, design of primers
According to the L2 gene order (accession number 132566356) of 4 type BTV listed in Genbank.Utilize albumen point
It analyses software etc. and analysis and prediction is carried out to L2 gene, the L2 gene of 4 types is divided into 3 segments A, B, C: 4A:1-1203bp；4B:
976-1980bp；4C:1861-2871bp；Design PCR amplification primer, 4A (P3-P6), 4B (P7-P10), 4C (P11-P14)
PCR amplification primer sequence as shown in the following table 1, table 2, table 1 be for pET-28a-c (+) carrier primer sequence, table 2 be for
The primer sequence of pMAL-c5x carrier:
Table 1 is directed to the primer sequence of pET-28a-c (+) carrier
Table 2 is directed to the primer sequence of pMAL-c5x carrier
2, gene magnification
Using the BTV4L2 gene of synthesis as template, PCR is carried out to the segment 4B using specific upstream and downstream primer pair P7-P8
Amplification.Reaction system is as follows:
PCR instrument is expanded after mixing, amplification condition are as follows: enters circulation, loop parameter 94 after 94 DEG C of 5min of initial denaturation
DEG C 1min, 53 DEG C of 40s, 72 DEG C of 1min, after 30 circulations, 72 DEG C of extensions 10min, 4 DEG C are saved.The PCR product of acquisition is passed through
0.8% agarose gel electrophoresis qualification result.
Other segments are expanded according to above-mentioned system and PCR amplification condition, and what is expanded respectively arrives for pET-28a-c
3 segments 4A, 4B, 4C of (+) carrier and pMAL-c5x vector construction.
3, the building of 4BTV-VP2 albumen pronucleus expression recombinant vector
PCR product and l0 × Loading Buffer are mixed, after 1.0% agarose gel electrophoresis, shone in ultraviolet lamp
It penetrates down and cuts the gel containing target fragment, place it in the EP pipe weighed, the weight of gel is calculated, according to a small amount of
The operation of DNA plastic recovery kit method: 300 μ L Buffer S1 are added in every l00mg nucleic acid glue, are placed in molten in 56 DEG C of water-baths
Solution is mixed by inversion once every 2-3min concussion in the process, when it melts completely, according to 150 μ L isopropanols/l00mg gel
Volume is added isopropanol and mixes, and gained liquid is added in adsorption column, and 12000rpm is centrifuged 1min, discards filtrate.It is added 700
For μ L rinsing liquid in adsorption column, 12000rpm is centrifuged 1min, discards filtrate.It is primary to add 600 μ L rinsing liquids repeated washing,
12000rpm centrifugation 1min discards filtrate.Repeat above-mentioned regulation, sky centrifugation 2min.Going for 40 μ L preheating is added in adsorbed film center
Ionized water, staticaccelerator adsorption 2min, 12000rpm are centrifuged 2min, collect eluent in new centrifuge tube.PCR after purification is produced
Object is connect with pMD18-T simple carrier, and is transferred in TG1.
Target gene 4A, 4B, 4C after purification is attached with carrier pET-28a-c (+), pMAL-c5x respectively.
Connection reaction total system is 10 μ L:Solution, I 5 μ L, pMD18-T simple carrier, 0.3 μ L, purification and recovery
4.7 μ L of PCR product.It is put into after mixing in connection instrument, 16 DEG C of connection 4h.
4, convert and choose bacterium
Product after connection is transformed into competent escherichia coli cell TG1: it is thin to take out competence from -80 DEG C of refrigerators
Born of the same parents are placed on ice to melt 3min, and 10 μ L of product after connection is added in 100 μ L of TG1, mix, stand 20min on ice.42℃
Thermal shock 100s, ice bath 5min.LB liquid recovery media 700 μ L, 37 DEG C of shaking table shaken cultivation 40min are added thereto.It takes out
Bacterium solution is spread evenly across on the LB plate containing Amp+ after 200 μ L culture, is all cultured after base absorbs after bacterium solution and is put into 37 DEG C
In incubator, it is inverted culture 12h.
5, the digestion identification and sequencing of recombinant expression plasmid
Recombinant plasmid is extracted with the small extraction reagent kit of plasmid.Bacterium solution after taking 1mL to cultivate is in EP pipe, 12000rpm centrifugation
1min discards supernatant, and 250 μ L Buffer P1 are added and mixing, which fullys shake, makes bacterial sediment suspend sufficiently；Add 250
μ L Buffer P2, mild turning upside down mix 6-8 times；350 μ L Buffer P3 are added, mild turning upside down mixes 5-
6 times, 12000rpm is centrifuged 10min；Gained supernatant is added in adsorption column, 12000rpm is centrifuged 1min, discards filtrate.Add
Enter 700 μ L rinsing liquids in adsorption column, 12000rpm is centrifuged 1min, discards filtrate.Add 500 μ L rinsing liquids repeated washing one
Secondary, 12000rpm is centrifuged 1min, discards filtrate, sky centrifugation 2min.Adsorption column is put into a new EP pipe, in adsorbed film
The deionized water of 60 μ L preheating is added in centre, and staticaccelerator adsorption 2min, 12000rpm are centrifuged 1min, collect Plasmid DNA eluent in new
Centrifuge tube in.
The plasmid of extraction carries out single double digestion identification, and 37 DEG C of water-baths act on 30min.Product is through 0.8% fine jade after single double digestion
Sepharose electroresis appraisal, as a result as shown in Figures 1 and 2.Digestion is identified that correct positive colony plasmid is sent to Suzhou gold only
Intelligence Biotechnology Co., Ltd is sequenced, and it is completely correct to compare objective gene sequence.The recombinant expression plasmid that building is obtained
It is respectively designated as pET-28a (+)-VP2-4A, pET-28a (+)-VP2-4B, pET-28a (+)-VP2-4C, pMAL-c5x-VP2-
4A, pMAL-c5x-VP2-4B and pMAL-c5x-VP2-4C.
6, the prokaryotic expression of BTV4-VP2 gene and purifying
According to 4 method for transformation by positive recombinant plasmid pET-28a (+)-VP2-4A, pET-28a (+)-VP2-4B,
PET-28a (+)-VP2-4C, pMAL-c5x-VP2-4A, pMAL-c5x-VP2-4B and pMAL-c5x-VP2-4C are converted respectively
To prokaryotic expression BL21 competent cell, then takes out 100 μ L bacterium solutions and be coated on containing ammonia section penicillin (100 μ g/mL)
On LB plate, 37 DEG C of overnight incubations, the white on picking LB plating medium isolates bacterium colony, is inoculated in 5mL and contains ammonia section mould
37 DEG C of overnight incubations in the LB liquid medium of plain (100 μ g/mL).Induced expression concrete operations are as follows: taking 1mL recombinant bacterium bacterium solution
When being added to that 37 DEG C of shake cultures are about 0.5-1 to OD600nm in the LB culture medium of 100mL, add IPTG to final concentration of
1mmol/L, 37 DEG C of induction 4-5h.Expression product is subjected to SDS-PAGE electrophoresis, is purified by cutting gluing method.
Expression vector pET-28a identifies with the target gene 4B recombinant bacterium constructed through SDS-PAGE, the recombinant bacterium after induction
Occur specific band at 46kD respectively, be consistent with expected size, and its expression-form is inclusion body expression, as a result such as Fig. 3
Shown, His-4B purifying protein and the Western blot analysis result of His label monoclonal antibody are as shown in Figure 5；Expression vector pMAL-
C5x identifies that the recombinant bacterium after induction occurs at 80kD special respectively with the target gene 4B recombinant bacterium constructed through SDS-PAGE
Property band, be consistent with expected size, and predominantly solubility expression, as a result as shown in Figure 4.
After recombinant bacterium pMAL-c5x-VP2-4B/BL21 is largely induced, carried out using the affinity column of GST label protein
Purifying.Albumen after purification measures protein concentration, -80 DEG C of cryopreservations after packing using micro-spectrophotometer.
Recombinant bacterium pET-28a (+)-VP2-4B/BL21 is largely induced, inclusion body is extracted.By cutting glue, electroelution
Mode albumen is purified.Albumen after purification, with the concentration of micro-spectrophotometer detection albumen, with -80 after packing
DEG C cryopreservation.
VP2-4A and VP2-4C albumen is identified and is purified according to processing method identical with VP2-4B albumen.
The preparation of 2 monoclonal antibody of embodiment
1, mouse immune
Mouse immune is to cut the recombination BTV4-VP2-4B egg of pET-28a (+)-VP2-4B/BL21 prokaryotic expression of glue purification
It is white that 4 4-6 week old female BAl BIc/C mices are immunized, it is immunized 3 times altogether, each immunization interval two weeks, immunizing dose is 50 μ g/,
Equivalent Freund's complete adjuvant and protein emulsifying are used for the first time, take equivalent incomplete Freund's adjuvant to be emulsified for the second time, for the third time,
Immunization route is peritoneal immunity.Booster immunization before merging.
2, cell fusion
First 1 day preparation feeder cells are merged, BALB/C mice peritoneal macrophage is conventionally taken to be laid on 96 holes
It is stand-by in tissue culture plate.Eyeball blood sampling, separates serum keeping, puts to death the mouse of spleen to be taken, sterile to take spleen and separating Morr. cell,
Cell fusion is carried out with PEG in the ratio of splenocyte and SP2/0 myeloma cell 8:1, fused cell is laid on ready
On feeder cells.
3, the screening of positive hybridoma cell strain and clone
Utilize the BTV4-VP2-4B egg with MBP label of pMAL-c5x-VP2-4B/BL21 prokaryotic expression after purification
It is white to establish indirect ELISA detection method screening positive hybridoma cell strain, culture is expanded to the hybridoma of reacting positive, together
When the subclone of positive hybridoma cell is carried out with limiting dilution assay, be at least subcloned 3 times, the positive hybridoma that will be subcloned
Cell freezes in time.It is final obtain one plant can with the hybridoma cell strain of the anti-BTV4-VP2 protein monoclonal antibody of stably excreting,
And the monoclonal antibody secreted is named as hybridoma cell strain 1B6, which is characterized in that the hybridoma cell strain preservation
In China typical culture collection center, address is in Wuhan University, microbial preservation number are as follows: CCTCC No.C2016107.
The identification of 3 monoclonal antibody of embodiment
1, the subgroup identification of monoclonal antibody
It is identified through Subclass of antibody identification kit, in the monoclonal antibody of anti-BTV4VP2 albumen, the subclass of 1B6 is IgG1.
2, Westernblot is tested
The atopic of monoclonal antibody is identified by Western blot test.By pMAL-c5x-VP2-
The recombination 4VP2-B albumen of 4B/BL21 expression is transferred on NC film, using positive hybridoma cell strain 1B6 culture supernatant as one
Anti-, the mountain sheep anti-mouse igg of HRP label is as secondary antibody, ECL colour developing.
Test result confirms that monoclonal antibody 1B6 prepared by the present invention can occur special with BTV4-VP2-B albumen
Property reaction, and (such as Fig. 6) is not reacted with BTV4-VP2-A, BTV4-VP2-C.
3, Sandwich ELISA detection monoclonal antibody 1B6 and BTV are viral reacts
The coated elisa plate of rabbit-anti BTV serum, every hole 100 μ L, 37 DEG C of incubation 2h.PBST is washed three times, each 5min.Often
50 μ L BTV-4 type virus of Kong Zhongjia, 37 DEG C of 1h.Board-washing 3 times.1:5 dilutes 1B6 supernatant, every 50 μ L of hole.37℃1h；Board-washing 3 times.
1:8000 dilutes sheep anti mouse secondary antibody (HRP), every 50 μ L of hole.37 DEG C 1 hour；Board-washing 3 times.TMB develops the color 15 minutes, adds terminate liquid,
Readings, the results are shown in Table 3.
3 Sandwich ELISA of table detection monoclonal antibody 1B6 is reacted with BTV virus
| 1 (BTV-4 type virus) | 2 (BTV-8 type viruses) | |
| 1B6 | 0.5912 | 0.2564 |
| Positive control | 1.7163 | 1.6333 |
| Negative control | 0.1294 | 0.179 |
Test result confirms, with BTV-4 type virus have it is certain react, and do not react with BTV-8 type virus.
Claims (4)
1. the hybridoma cell strain of one plant of anti-BTV4-VP2 protein monoclonal antibody of secretion, is named as hybridoma cell strain 1B6,
It is characterized in that, the hybridoma cell strain is deposited in China typical culture collection center, microbial preservation number are as follows:
CCTCC No.C2016107。
2. the monoclonal antibody secreted by hybridoma cell strain described in claim 1.
3. application of the hybridoma cell strain described in claim 1 in preparation detection 4 type reagent of blue tongue virus serum.
4. application of the monoclonal antibody as claimed in claim 2 in preparation detection 4 type reagent of blue tongue virus serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610533234.1A CN106190986B (en) | 2016-07-07 | 2016-07-07 | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610533234.1A CN106190986B (en) | 2016-07-07 | 2016-07-07 | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106190986A CN106190986A (en) | 2016-12-07 |
| CN106190986B true CN106190986B (en) | 2019-05-17 |
Family
ID=57472636
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610533234.1A Active CN106190986B (en) | 2016-07-07 | 2016-07-07 | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106190986B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107082802B (en) * | 2017-03-01 | 2020-02-11 | 东北农业大学 | Neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof |
| CN107179408B (en) * | 2017-05-08 | 2019-01-22 | 东北农业大学 | Competitive ELISA detection kit and its application for the detection of blue tongue virus type specificity |
| CN107271667B (en) * | 2017-06-05 | 2019-01-22 | 东北农业大学 | Blue tongue virus type specificity competitive ELISA detection kit and its application |
| CN110229791A (en) * | 2018-03-05 | 2019-09-13 | 云南省畜牧兽医科学院 | A kind of hybridoma for secreting anti-blue tongue virus NS3 protein monoclonal antibody |
| CN117801099B (en) * | 2023-12-11 | 2024-08-30 | 中国农业科学院兰州兽医研究所 | Recombinant monoclonal antibody of anti-bluetongue virus VP7 protein |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403759A (en) * | 2008-11-13 | 2009-04-08 | 中国人民解放军军事医学科学院野战输血研究所 | ELISA detection method and reagent kit for bluetongue viral antigen |
| CN101597334A (en) * | 2009-07-23 | 2009-12-09 | 花群义 | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application |
| CN102776152A (en) * | 2012-06-26 | 2012-11-14 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody against BTV (bluetongue virus) VP7 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain |
| CN103305470A (en) * | 2013-06-18 | 2013-09-18 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody BTV8-VP2-3E11 resistant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application |
| CN103642757A (en) * | 2013-10-14 | 2014-03-19 | 中国农业科学院哈尔滨兽医研究所 | Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications |
| CN105255837A (en) * | 2015-10-16 | 2016-01-20 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application |
-
2016
- 2016-07-07 CN CN201610533234.1A patent/CN106190986B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403759A (en) * | 2008-11-13 | 2009-04-08 | 中国人民解放军军事医学科学院野战输血研究所 | ELISA detection method and reagent kit for bluetongue viral antigen |
| CN101597334A (en) * | 2009-07-23 | 2009-12-09 | 花群义 | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application |
| CN102776152A (en) * | 2012-06-26 | 2012-11-14 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody against BTV (bluetongue virus) VP7 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain |
| CN103305470A (en) * | 2013-06-18 | 2013-09-18 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody BTV8-VP2-3E11 resistant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application |
| CN103642757A (en) * | 2013-10-14 | 2014-03-19 | 中国农业科学院哈尔滨兽医研究所 | Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications |
| CN105255837A (en) * | 2015-10-16 | 2016-01-20 | 中国农业科学院哈尔滨兽医研究所 | Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application |
Non-Patent Citations (3)
| Title |
|---|
| 25型蓝舌病病毒VP7和VP2蛋白的原核表达及其单克隆抗体的制备;武啸;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160415;全文 * |
| 蓝舌病病毒15型VP2蛋白单克隆抗体的制备及其抗原表位鉴定;张沁等;《中国预防兽医学报》;20150531;第37卷(第5期);全文 * |
| 蓝舌病病毒4型VP5蛋白单克隆开抗体制备及其抗原表位的鉴定;孙亮;《中国优秀硕士学位论文全文数据库 农业科技辑》;20141115;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106190986A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106190986B (en) | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody | |
| Sánchez-Fauquier et al. | Characterization of a human astrovirus serotype 2 structural protein (VP26) that contains an epitope involved in virus neutralization | |
| CN107099506A (en) | Porcine epidemic diarrhea virus double-antibody sandwich elisa immue quantitative detection reagent box and its application | |
| CN103756973B (en) | A kind of indirect ELISA testing kit of GCRV | |
| CN116836270B (en) | Monoclonal antibody of anti-bluetongue virus VP7 protein, preparation method and application | |
| CN106380515B (en) | Hybridoma cell strain capable of stably secreting anti-PEDV monoclonal antibody, antibody secreted by hybridoma cell strain and application of hybridoma cell strain | |
| Hoshino et al. | An equine rotavirus (FI-14 strain) which bears both subgroup I and subgroup II specificities on its VP6 | |
| CN108329378A (en) | Senecan paddy virus VP 1 albumen, encoding gene, hybridoma cell strain and monoclonal antibody and its application | |
| Liu et al. | Neutralization of genotype 2 porcine epidemic diarrhea virus strains by a novel monoclonal antibody | |
| CN101597334A (en) | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application | |
| CN104371980B (en) | A kind of Sabin strains polio type III viral monoclonal antibodies and its application | |
| CN104498439B (en) | Hybridoma, PCV2 monoclonal antibodies and its application | |
| CN113817687B (en) | Hybridoma cell strain, influenza A virus nucleoprotein monoclonal antibody and application thereof | |
| CN111999497A (en) | Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application thereof | |
| CN103740649B (en) | The B cell epi-position of anti-blue tongue virus serum 16 type VP2 protein monoclonal antibody BTV16-2B4 and identification thereof and application | |
| CN107312088A (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kits and its application | |
| CN109959788B (en) | Coating antigen and enzyme-labeled antibody for detecting porcine pseudorabies virus gE antibody and blocking method kit | |
| CN101322844B (en) | Non-virus non-structural protein foot-and-mouth disease vaccine and preparation thereof | |
| CN104357400A (en) | Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof | |
| CN102229915A (en) | EV71 virus monoclonal antibody, hybridoma cell line and application | |
| CN104371979A (en) | Sabin strain poliovirus type II monoclonal antibody and application thereof | |
| CN102532310B (en) | Monoclonal antibody against grass carp reovirus VP5 protein and application of monoclonal antibody | |
| CN105177016A (en) | Anti-chloramphenicol-scFv antibody gene and application thereof | |
| CN107058236A (en) | A kind of hybridoma cell strain and its anti-Aleutian Mink Disease Parvovirus monoclonal antibody of generation | |
| CN107271667A (en) | Blue tongue virus type specificity competitive ELISA detection kit and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |