CN103088033B - The antibody gene of anti-pyrethroid pesticide scFv and application - Google Patents
The antibody gene of anti-pyrethroid pesticide scFv and application Download PDFInfo
- Publication number
- CN103088033B CN103088033B CN201310029812.4A CN201310029812A CN103088033B CN 103088033 B CN103088033 B CN 103088033B CN 201310029812 A CN201310029812 A CN 201310029812A CN 103088033 B CN103088033 B CN 103088033B
- Authority
- CN
- China
- Prior art keywords
- antibody
- scfv
- antibody gene
- gene
- variable region
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 48
- 239000002728 pyrethroid Substances 0.000 title abstract description 34
- 239000000575 pesticide Substances 0.000 title abstract description 29
- 150000001413 amino acids Chemical class 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 18
- NYPJDWWKZLNGGM-UHFFFAOYSA-N fenvalerate Chemical compound C=1C=C(Cl)C=CC=1C(C(C)C)C(=O)OC(C#N)C(C=1)=CC=CC=1OC1=CC=CC=C1 NYPJDWWKZLNGGM-UHFFFAOYSA-N 0.000 claims description 14
- 238000010353 genetic engineering Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 abstract description 10
- 238000002823 phage display Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 3
- 108091007433 antigens Proteins 0.000 abstract description 3
- 238000012163 sequencing technique Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 19
- 238000000034 method Methods 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 14
- 238000011534 incubation Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 230000005611 electricity Effects 0.000 description 5
- 239000003292 glue Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 238000011530 RNeasy Mini Kit Methods 0.000 description 3
- 239000003905 agrochemical Substances 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 244000124209 Crocus sativus Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108060003951 Immunoglobulin 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
- 229920000297 Rayon Polymers 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- VEMKTZHHVJILDY-UXHICEINSA-N bioresmethrin Chemical compound CC1(C)[C@H](C=C(C)C)[C@H]1C(=O)OCC1=COC(CC=2C=CC=CC=2)=C1 VEMKTZHHVJILDY-UXHICEINSA-N 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000007681 cardiovascular toxicity Effects 0.000 description 1
- 231100000060 cardiovascular toxicity Toxicity 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 230000005264 electron capture Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 108010063039 endodeoxyribonuclease SfiI Proteins 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 150000002314 glycerols Chemical class 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Abstract
The present invention relates to the antibody gene of a kind of anti-pyrethroid pesticide scFv, relate to the structure of pyrethroid pesticide antibody library, the screening of specific single-chain antibody gene, the sequencing of specific single-chain antibody gene, the preparation of specific phage display single-chain antibody, this antibody gene comprises encoding heavy chain variable region (116 amino acid), the nucleotide sequence of flexible link zone (15 amino acid) and variable region of light chain (107 amino acid), as shown in sequence table SEQ ID No.1 and SEQ ID No.2, and utilize phagemid to prepare in host cell to be illustrated in this antibody of phage surface form.And this antibody capable is at phage surface normal expression and folding, can identify pyrethroid antigen preferably.The present invention is compared with conventional antibodies, and this antibody has the advantages such as stability is high, easy to prepare, has important potential using value.
Description
Technical field
The present invention relates to gene engineering technology field and field of immunological detection, be specifically related to antibody gene and the application of a kind of anti-pyrethroid pesticide scFv.
Background technology
Pyrethroid (Pyrethroids) agricultural chemicals is the important biomimetic insecticide of a class, and the past is considered to a kind of safe agricultural chemicals always.Because it has the degraded of easy oxidized enzyme system in vivo, without the advantage such as accumulative, application is at present general, large usage quantity, accounts for 20% of insecticidal total output, accounts for 25% of whole sterilant usable floor area.But there are some researches show recently, major part pyrethroid pesticide belongs to environmental hormone class material, there is immunity system toxicity, genetoxic and Cardiovascular Toxicity, have a strong impact on the normal physiological activity of mammal, even low dosage, Long Term Contact also easily causes chronic disease, and some also has the effects such as teratogenesis, carcinogenic, mutagenesis.Therefore, the residual problem having become people's extensive concern of this agricultural chemicals in agricultural-food how is effectively removed.
Because the content of this kind of pesticide residue is atomic, therefore require super-sensitive detection technique.The pyrethroid pesticide remained quantity measuring method reported has gas-chromatography (GC) method, Enzyme-linked Immunosorbent Assay (ELISA) etc.GC method uses highly sensitive electron capture detector, thus requires strict to the purification of sample, and adds the analysis process time.This just urgently needs a kind of method of new rapid detection; Though common euzymelinked immunosorbent assay (ELISA) is highly sensitive, sample capacity is large, have that laboratory animal requirement is large, breeding cycle is long, different animals is individual to agricultural and veterinary chemicals sensitivity difference, causes the problems such as antibody variability is large; Monoclonal antibody as second generation antibody, due to preparation and screening process loaded down with trivial details, and require very high to operative technique, the cytogenetics after clone is the reason such as instability easily, is unfavorable for widespread use.
The Heterosis of genetic engineering antibody is easy in saving laboratory animal and feeding time, technology, be easy to put into production, genetic information has operability.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of with strong points, scFv antibody of anti-pyrethroid pesticide that stability is high, easy to prepare and the gene order of this antibody of encoding are provided.
It take pyrethroid pesticide as the high abundance scFv antibody library preparation method detecting target that another object of the present invention is to provide a kind of.
Present invention also offers a kind of is efficiently the screening method of the scFv antibody library of target for pyrethroid pesticide.
The technical scheme that the present invention realizes object is as follows:
A heavy chain gene for the antibody gene of anti-pyrethroid pesticide scFv, the sequence of described heavy chain gene is shown in sequence 1.
A light chain gene for the antibody gene of anti-pyrethroid pesticide scFv, the gene order of light chain gene is shown in sequence 2.
An antibody gene of anti-pyrethroid pesticide scFv, it comprises heavy chain gene and light chain gene, and this gene is made up of 714 Nucleotide, and the sequence of described heavy chain gene is shown in sequence 1, and the gene order of light chain gene is shown in sequence 2.
The antibody of a kind of anti-pyrethroid pesticide scFv encoded by the antibody gene of anti-pyrethroid pesticide scFv.
And variable region of heavy chain is made up of 116 amino acid, flexible link zone is by 15 amino acid, and variable region of light chain is made up of 107 amino acid.
Change, lack or add one or several amino acids formed scFv antibody with same function through spy by the aminoacid sequence of scFv antibody.
The carrier of the antibody gene containing anti-pyrethroid pesticide scFv.
The genetic engineering bacterium of the antibody gene containing anti-pyrethroid pesticide scFv.
The application that the antibody gene of anti-pyrethroid pesticide scFv detects at pyrethroid pesticide and prepares in anti-pyrethroid scFv antibody.
The application of genetic engineering bacterium in the anti-pyrethroid scFv antibody of preparation of the antibody gene of anti-pyrethroid pesticide scFv.
Advantage of the present invention and positively effect as follows:
The present invention has prepared the scFv antibody of the phage display of anti-pyrethroid pesticide, PCR-based technology and phage display technique, and the present invention has the following advantages:
(1) the application is by consulting a large amount of NCBI design data degenerate primer, this primer can cover the diversity of mouse source heavy chain of antibody and light chain all sidedly, especially light chain primer is except comprising general kappa light chain primer, further comprises lambda light chain primer, with the loss of the gene of preventing portion subhead;
(2) the present invention selects hybridoma as the gene source of the anti-pyrethroid scFv antibody of preparation, eliminate immunity, that feeding animals brings is loaded down with trivial details, specific aim is stronger, also eliminate the interference of a large amount of non-object antibody brought with the direct elutriation of mouse spleen immune library simultaneously, decrease blindness;
(3) because phage antibody diluent comprises a certain proportion of carrier proteins and confining liquid composition, in the affine adsorption step of elutriation, the application optimizes phage antibody dilution buffer, to be conducive to the interference that screening antibodies is more targeted, eliminating carrier proteins, confining liquid bring;
(4) the preparation method of the application saves time, technology is easy, be easy to that development downstream produces, genetic information can operate and stable.
Accompanying drawing explanation
Fig. 1: take PBA-cDNA as template, pcr amplification heavy chain of antibody, light chain figure.In figure: M:DL2000 nucleic acid molecular weight Marker, VH: heavy chain of antibody, VL: light chain of antibody.
Fig. 2: carry out Overlap extension PCR with heavy chain, light chain and flexible peptide linker, obtains complete scFv antibody.M:DL2000 nucleic acid molecular weight Marker, scFv: complete single-chain antibody.
Embodiment
Below in conjunction with embodiment, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
In order to realize the object of the invention, applicant utilizes recombinant DNA technology and display technique of bacteriophage, heavy chain of antibody, the chain variable region gene for pyrethroid pesticide with the mouse source hybridoma cell strain 2G2E7 of Broadspectrum specificity are carried out amplification in vitro, again through Overlap extension PCR, with 15 amino acid (aminoacid sequence (GGGGS)
3, gene order GGTGGAGGCG GTTC AGGCGGAGGTGGCTCTGGCGGTGGCGGATCG)) heavy chain, light chain connect into scFv antibody by the flexible peptide linker that forms.Then the scFv gene of gained is inserted in pCANTAB-5E Vector for Phage Display, construction recombination plasmid electricity is converted in Host Strains, obtains the antibody library of a cell derived.Take turns stationary phase elutriation and qualification through 5, obtain the phage display scFv antibody of anti-pyrethroid pesticide.It heavy chain comprised and light chain gene sequence are respectively as SEQ ID No.1 and SEQ ID No.2.
Present invention also offers scFv heavy chain, light chain aminoacid sequence as sequence 18 and sequence 19.
Present invention also offers the degenerate primer by NCBI design, for the said gene that increases.Wherein light chain amplimer also introduces lambda strand primer, to cover the diversity of antibody variable region except conventional kappa primer.
Present invention also offers the host cell of carrier containing said gene and carrier.Present invention also offers above-mentioned scFv antibody, the gene of this antibody of encoding, the carrier containing this gene, the application of the host cell containing this carrier in the anti-pyrethroid pesticide scFv antibody of preparation.When present invention also offers elutriation antibody library, process phage antibody used, through optimize remove interferenceization treatment solution, to strengthen the specific aim of elutriation.
Experimental technique in following examples, if no special instructions, is ordinary method.
The reagent material used in following examples, if no special instructions, is the purchase of routine biochemistry reagent suppliers and obtains.
In following examples due to pyrethroid pesticide universal hapten when synthesizing referred to as PBA, so all referred to as PBA when following corresponding cell, gene, bacterial classification etc. mention relevant pyrethroid pesticide.
The extraction of embodiment 1PBA monoclonal cell total serum IgE and the synthesis of cDNA
1, the extraction of PBA monoclonal cell strain 2G2E7 total serum IgE
Get the fresh monoclonal cell strain 2G2E7 obtained by this laboratory screening, utilize Qigen Rneasy Mini Kit to carry total serum IgE, cell strain 2G2E7 can produce the monoclonal antibody of anti-PBA.
(1) culturing cell is to 3-4 × 10
6individual, the centrifugal 5min of 300 × g, removes supernatant;
(2) prepare lysate: Buffer RLT: beta-mercaptoethanol=1ml:10 μ l;
(3) in the cell after centrifugal, add 350 μ l Buffer RLT lysis buffers, flick tube wall, with liquid-transfering gun piping and druming, damping fluid is fully contacted with cell;
(4) add the ethanolic soln of 70% of equal volume, liquid-transfering gun is inhaled gently and is played mixing, notes not centrifugal;
(5) get the silicagel column in Qigen Rneasy Mini Kit, load on 2ml collection tube, shift all samples in pillar, under room temperature, the centrifugal 15s of 12000rpm, discards waste liquid;
(6) add 700 μ l Buffer RW1 in post, under room temperature, the centrifugal 15s of 12000rpm, discards effluent liquid;
(7) add the RPE damping fluid of 500 μ l alcohol dilutions, washing pillar, 10000rpm, centrifugal 15s, abandons supernatant;
(8) add 500 μ l RPE damping fluids and move into pillar, froth lid, 10000rpm under room temperature, centrifugal 2min, rinse pillar;
(9) pillar is put into the new 2ml collection tube that a test kit provides, at full speed centrifugal 1min;
(10) in 1.5ml centrifuge tube posts transfer provided to Qigen Rneasy Mini Kit, that gets 50 μ l is added drop-wise on the film of pillar without RNA enzyme water (test kit provides) is carefully unsettled, leaves standstill 3-5min, gently cover lid, the centrifugal 1min of 10000rpm under room temperature, eluted rna;
Wash-out RNA packing, mark after, frozen in-80 DEG C.
2, the synthesis of PBA-cDNA
(1) add following component to 1.5ml centrifuge tube:
(2) get togather lid, centrifuge tube is put into 70 DEG C of water-bath 5min, be then put into immediately on ice, at least 5min, centrifugal 10s collects enriched material and keeps initial volume, puts on ice always;
(3) prepare reverse transcription system, each component is put on ice always;
(4) the reaction system that previous step prepares mixes with the reaction system of the first step, final volume 20 μ l;
(5) anneal: 25 DEG C, 5min;
(6) extend: 42 DEG C, 1h;
(7) packing ,-80 DEG C frozen.
The structure of embodiment 2PBA-scFv single-chain antibody library
1, pcr amplification VH, VL
The primer: (after VL, after primer and scFv, primer is that primer is equivalent in use and uses)
Wherein K is kappa primer, and L is lambda primer, also to increase lambda chain, so primer before not having independent lambda with primer before kappa.
PCR system (50 μ l):
PCR program:
Through pcr amplification, product utilization agarose gel electrophoresis detects, and result as shown in Figure 1.
2, Qiagen gel purification kit glue is utilized to reclaim object fragment
(1) prepare clean centrifuge tube, weigh;
(2) cut the object fragment on sepharose with clean blade, put into centrifuge tube;
(3) weigh, calculate the glue weight cut, add the Buffer QG of 3 times of volumes in 1 times of volume gel;
(4) hatch for 50 DEG C, 10min, until glue dissolves completely, put upside down once every 2-3min;
(5), after glue dissolves completely, check whether solution colour is yellow, as solution colour becomes safran or purple, add the mixing of 10ul3M sodium-acetate;
(6) double gel volume Virahol in solution, mixing (100mg-100ul Virahol);
(7) the silicon gel column in test kit is put into 2ml collection tube;
(8) in conjunction with DNA: be added to by solution on pillar, centrifugal 1min, 13000rpm(liquor capacity is once centrifugal again more than 800ul);
(9) use up effluent liquid, pillar is put back in centrifuge tube again;
(10) add 0.5ml Buffer QG on pillar, centrifugal 1min, 13000rpm;
(11) wash-out: use up effluent liquid, add 750ul Buffer PE on pillar, centrifugal 1min;
(11) use up effluent liquid, centrifugal 1min, 13000rpm;
(12) pillar is put into a clean 1.5ml centrifuge tube;
(13) elution DNA, adding 50ul Buffer EB or pH is in the middle of 7.0-8.5 water to film, centrifugal 1min;
3, eppendorf Bio-PhotoMeter plus is utilized to measure VH and VL concentration
4, PBA-scFv synthesis
A. pre-splicing system:
PCR program:
Gained PCR primer is used for the formal splicing of scFv.
B.scFv formally splices system
PCR program:
Through pcr amplification, product agarose gel electrophoresis detects, and the results are shown in Figure 2.Cut the object band of about about 750bp, reclaim with Qiagen gel purification kit glue, eppendorf Bio-PhotoMeter plus measures concentration ,-20 DEG C of preservations.
5, antibody library builds
A. double digestion is carried out to pCANTAB-5E carrier and scFv
Get the pCANTAB-5E carrier prepared, first build sfiI endonuclease reaction system:
Reaction system is put into 50 DEG C of water-bath enzymes and cut 4h.
Be cooled to room temperature, then cut with NotI enzyme, namely add 1 μ lNotI, fully put into 37 DEG C of water-bath enzymes after mixing and cut 4h.Then get plasmid enzyme restriction product to mix with Loading Buffer, join in 1% sepharose, 120V electrophoresis, under ultraviolet, observe the object band whether having and meet size (about 4.5kb).
Reclaimed by the digested plasmid blob of viscose Qiagen gel purification kit cut, scFv fragment uses QiagenPCR purifying to reclaim test kit and reclaims.The carrier reclaimed and scFv fragment eppendorf Bio-PhotoMeter plus are measured concentration.
B. connect
Use the T of Promega
4scFv fragment after cutting through sfiI with NotI enzyme is connected with pCANTAB-5E carrier by ligase enzyme.Working method is as the T of Promega
4enzyme specification sheets.
Get the pCANTAB-5E carrier after appropriate purifying and scFv fragment, build ligation system:
Linked system is placed in 16 DEG C of water-baths to gather, connects 16h.
C. electricity transforms the preparation of TG1 competent cell
(1) from-80 DEG C of refrigerators, take out t bacteria G1, line on 2 × YT flat board, cultivate 16-20h for 37 DEG C;
(2) the single colony inoculation of picking is in 5ml2 × YT, 37 DEG C, and 180rpm shakes overnight incubation;
(3) getting the bacterium liquid 500 μ l that spends the night is inoculated in 50ml2 × YT nutrient solution, 37 DEG C, and 180rpm concussion is cultured to OD600 and reaches 0.7, and bacterium liquid puts into mixture of ice and water;
(4) be then transferred in precooling centrifuge tube, 4 DEG C, the centrifugal 10min of 2000 × g, obtain bacterium precipitation;
(5) with the ddw gently outstanding bacterial sediment of precooling, 4 DEG C, 2000 × g, centrifugal 10min, abandons supernatant;
(6) repeat above step e twice, 2400 × g with 10% glycerine, centrifugal 10min, to the greatest extent supernatant, to suspend thalline with glycerine, packing centrifuge tube, 100 μ l/ manage;
(7) the bacterium liquid of packing is put into liquid nitrogen quick-frozen ,-80 DEG C of preservations.
D. electricity transforms
(1) the competent cell that taking-up-80 DEG C is frozen, melts on mixture of ice and water.0.2cm gap electric shock cup is placed in precooling on ice simultaneously;
(2) get 100 μ l competent cells, add the connection product 10 μ l that step B obtains wherein, after mixing gently with pipettor, be placed in 90s on ice;
(3) added by mixed solution in the gap of precooling electric shock cup, electric shock cup is placed in electric conversion instrument, ensures that cup outer wall is dry;
(4) electricity turns optimum configurations and becomes 15kv/cm, 5ms, carries out electricity and turns;
(5) take out electric shock cup after electric shock, add the LB substratum of 1ml preheating immediately, gently after mixing, transfer conversion fluid is in 1.5ml sterile centrifugation tube, and 37 DEG C, 180rpm, 1.5h is cultivated in concussion.
6, Jian Ku
A. get the bacterium liquid 20 μ l that step cultivates 1.5h, do 2 times of dilutions with 2 × YT nutrient solution, coating 2 × YT-AG flat board (2 × YT containing ammonia benzyl and glucose is dull and stereotyped), is inverted overnight incubation for 30 DEG C.After colony growth, carry out plate count and calculate storage capacity.Remaining whole bacterium liquid, add 250 μ l80% sterile glycerols ,-80 DEG C frozen, and this is primary antibody storehouse.
B. the mono-clonal 10 random picking 2 × YT-AG flat board grown, extracts plasmid, with sfiI and NotI double digestion, calculates positive rate and identify whether also have false positive.Plasmid utilizes root plasmid little extraction reagent kit in sky to extract, and enzyme cuts step step as previously mentioned.As calculated, primary antibody Kuku holds for 2.3x10
7, positive rate is 95%.
The elutriation of embodiment 3PBA antibody library
1, prepare before elutriation
(1) get 1ml primary antibody storehouse, be added to 250ml2 × YT-G substratum, 37 DEG C, 1h is cultivated in 250rpm concussion;
(2), when bacterial growth to proper concn adds 100 μ g/ml AMP, the phage M13K07(stoste laboratory simultaneously adding the fresh preparation of moi=20:1 is preserved), 37 DEG C of water-bath jog 30min, afterwards 37 DEG C vibrate 250rpm, cultivate 30min;
(3) take out appropriate bacterium liquid and carry out 2 times of dilutions, coat respectively on 2 × YT-AG and 2 × YT-KG flat board (2 × YT containing ammonia benzyl and glucose is dull and stereotyped), determine Phage Infection success;
(4) the centrifugal 15min of 1000 × g, carefully gets supernatant;
(5) use 500ml2 × YT-AK suspended bacteria to precipitate, 37 DEG C, 250rpm overnight incubation;
(6) the centrifugal 20min of 12000rpm, gets supernatant in another new Tube, prepares to carry out elutriation.
2, PEG/Nacl precipitating phage
(1) in supernatant, add 100ml PEG/Nacl, precipitating phage 50min on 4 DEG C of ice baths;
(2) 4000rpm, 4 DEG C of centrifugal 20min, abandon supernatant, as far as possible removing tube wall raffinate;
(3) to suspend phages with 5mlPBS, the centrifugal 15min of 10000rpm, gets supernatant for subsequent use;
(4) get the phage supernatant that wherein 50 μ l obtain, 2 times of dilutions are done with 2 × YT substratum, respectively get diluent 100 μ l and be added to the TG1 Host Strains that OD600 is 0.5,37 DEG C of jogs hatch 20min, coating 2 × YT-AG is dull and stereotyped, 30 DEG C of overnight incubation, for calculating the input of phage, the phage supernatant obtained is immediately for elutriation.
3, elutriation
(1) elutriation was wrapped managed antigen coated for effective for immunity PBA-OVA by 2ml/, 10 μ g/ml the day before yesterday;
(2) turn sky PBS and wash pipe 3 times, pat dry;
(3) use 0.5%M-PBS(skimming milk-PBS) close immunity pipe, 37 DEG C of closed 2h;
(4) abandon confining liquid, PBS washes pipe 3 times, pats dry; Now, interferenceization treatment solution will be gone to join phage supernatant.Namely according to OVA-MPBS (final concentration is the PBS of 0.2wt%OVA, 0.3wt% milk powder): the volume ratio of supernatant=1:3, by the phage displaying antibody mixing of OVA, milk powder and acquisition, acts on 20min under room temperature, carries out interferenceization and process.
(5) add the phage supernatant that on 2ml/ pipe, step is handled well, jog incubation 30min, 37 DEG C of incubation 1.5h;
(6) abandon the phage displaying antibody added, PBST washs 3 times, and PBS washs 3 times, pats dry;
(7) add the Host Strains TG1 that OD600 is 0.5,2ml/ manages, 37 DEG C, 150rpm shaking culture 1h.Namely complete first round elutriation, obtain Primary antibodies storehouse.
(8) get the bacterium liquid of shaking culture, do 2 times of stepwise dilutions with 2 × YT substratum, get diluent 100 μ l and be coated with 2 × YT-AG flat board, 30 DEG C of overnight incubation, for calculating the phage work output of elutriation.
(9) in one-level storehouse, add the amp and 4 × 10 that final concentration is 2% glucose and 100 μ g/ml
10m13K07 phage.After leaving standstill incubation 30min, the centrifugal 10min of rear 250rpm shaking culture 30min, 1000 × g, abandons supernatant, and bacterium precipitation 100ml2 × YT-AK(contains 2 × YT nutrient solution of ammonia benzyl and kantlex) change liquid cultivation, 37 DEG C, 250rpm overnight incubation;
(10) the phage supernatant now obtained takes turns elutriation for second, and Panning methods is described above; The input of often taking turns and work output 2 × YT substratum do 2 times of stepwise dilutions, get diluent 100 μ l and are coated with 2 × YT-AG flat board, 30 DEG C of overnight incubation.
(11) eventually pass through 5 and take turns elutriation, the bacterium liquid of acquisition is by bacterium liquid: 80% sterile glycerol is the volume ratio mixing of 4:1, and-80 DEG C frozen, is 5 grades of antibody libraries.The plate clone of picking 5 grades of storehouse gained carries out PCR checking and digestion verification (method as previously mentioned), gets positive colony and carries out follow-up test.
Elutriation result:
Embodiment 4 indirect competition Phage-ELISA detects the positive recombinant antibodies of PBA
1, the expression of monoclonal phage recombinant antibodies in 5 grades of antibody libraries
A. get 1.5ml sterile centrifugation tube, add 2 × YT-AG substratum 500 μ l/ and manage;
B. choose step and demonstrate,prove 15 correct positive colonies, be seeded in the ready substratum of step;
C. be placed in shaking table, 30 DEG C, 200rpm, centrifuge tube inclination shaking culture is spent the night;
D. next day, get 500 μ l and contain 2.5 × 10
10in 2 × YT-AG to Amoxcillin 1.5ml centrifuge tube of pfu/ml M13K07;
E. from the mono-clonal of incubated overnight, every hole is got in the fresh culture of the supreme step preparation of 50 μ l nutrient solution;
F. shaking table is placed in, 37 DEG C, 150rpm, shaking culture 2 hours;
G.1500 the centrifugal 20min of × g, carefully removes supernatant;
H. in every pipe bacterium precipitation, add 500 μ l2 × YT-AK nutrient solutions, 37 DEG C, 250rpm, shaking culture is spent the night;
I.1500 the centrifugal 20min of × g, carefully gets supernatant stand-by.
2. Phage-ELISA
A. quilt is wrapped: with PBA-OVA coating antigen bag by 96 hole enzyme plates, 0.5 μ g/ hole, sets up positive control and negative control, and positive control now uses M13K07 to carry out bag quilt, 0.5 μ g/ hole;
B. wash: abandon coating buffer, wash 3 times with PBS, pat dry enzyme plate;
C. close: add 1% skimming milk PBS(MPBS) 200 μ l/ holes, 37 DEG C of closed 1.5h;
D. wash: abandon confining liquid, wash 4 times with PBS, pat dry enzyme plate;
E. add phage recombinant antibodies: the every hole of control group adds 50 μ lPBS in the enzyme mark hole closed, add the every hole of mark product group and add 50 μ l1ppm fenvalerate mark product.Afterwards, recombinant antibodies 50 μ l is added, 37 DEG C of association reaction 2h;
F. wash: abandon recombinant antibodies liquid, wash 4 times with PBST, then wash 4 times with PBS, pat dry enzyme plate;
G. ELIAS secondary antibody (purchase of Abcam company) is added: anti-for enzyme mark M13 antibody PBS is done 1:10000 dilution, 100 μ l/ holes, 37 DEG C of incubation reaction 1h;
H. wash: abandon enzyme labelled antibody liquid, wash 4 times with PBST, then wash 4 times with PBS, pat dry enzyme plate;
I. develop the color: add 50 μ l/ hole TMB and develop the color (purchase of sigma company), hatch colour developing 20min for 37 DEG C;
J. stop: with 200 μ l/ hole stop buffers, color development stopping, reads value at 450nm place.
Through Phage-ELISA, No. 12 clones demonstrate higher affinity and specificity.The phage recombinant antibodies supernatant that this clone produces is diluting 20 times with PBS, and during colour developing 20min, Positive control wells (wrapping by M13KO7) OD450nm light absorption value is 3.0.Control wells (do not add mark product, only add phage displaying antibody) OD450nm light absorption value is 1.210(n=3).The Phage-ELISA result of this clone is as follows:
3, positive colony order-checking
Get specificity in step Phage-ELISA result, good No. 12 clones of affinity deliver to Beijing Jin Weizhi company order-checking.Sequencing result shows, and the phage recombinant antibodies gene that this clone produces is immunoglobulin superfamily gene, namely obtains a kind of heavy chain and light chain gene of anti-pyrethroid pesticide single-chain antibody, as shown in SEQ.No1 and SEQ.No2.By DNAMAN software translation, the bright aminoacid sequence in result surface, without premature termination phenomenon, illustrates that recombinant antibodies is expressed successfully, can be illustrated in M13 phage surface and have the antibody activity of good pyrethroid pesticide.Utilize software translation, obtain the aminoacid sequence of heavy chain and light chain, respectively as shown in sequence 18 and sequence 19.Since then, a kind of antibody gene of anti-pyrethroid pesticide is namely obtained.
Claims (8)
1. the antibody gene of a Fenvalerate scFv, it is characterized in that: be made up of 714 Nucleotide of encoding heavy chain variable region, flexible link zone and variable region of light chain, the nucleotide sequence of encoding heavy chain variable region is as shown in SEQ ID NO:1, and the nucleotide sequence of encoded light chain variable region is as shown in SEQ ID NO:2.
2. the Fenvalerate scFv encoded by antibody gene described in claim 1.
3. Fenvalerate scFv according to claim 2, is characterized in that: variable region of heavy chain is made up of 116 amino acid, and flexible link zone is made up of 15 amino acid, and variable region of light chain is made up of 107 amino acid.
4. the carrier containing antibody gene according to claim 1.
5. the genetic engineering bacterium containing antibody gene according to claim 1.
6. the application of antibody gene according to claim 1 in fenvalerate detects.
7. antibody gene according to claim 1 is preparing the application in Fenvalerate scFv.
8. carrier according to claim 4 or genetic engineering bacterium according to claim 5 are preparing the application in Fenvalerate scFv.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310029812.4A CN103088033B (en) | 2013-01-25 | 2013-01-25 | The antibody gene of anti-pyrethroid pesticide scFv and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310029812.4A CN103088033B (en) | 2013-01-25 | 2013-01-25 | The antibody gene of anti-pyrethroid pesticide scFv and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103088033A CN103088033A (en) | 2013-05-08 |
| CN103088033B true CN103088033B (en) | 2015-09-16 |
Family
ID=48201163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310029812.4A Expired - Fee Related CN103088033B (en) | 2013-01-25 | 2013-01-25 | The antibody gene of anti-pyrethroid pesticide scFv and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103088033B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103724401B (en) * | 2014-01-08 | 2015-06-10 | 湖南文理学院 | Preparation method of peptide ligand for non-competitive detection of water-soluble pesticide residues |
| CN105177016A (en) * | 2015-09-17 | 2015-12-23 | 天津科技大学 | Anti-chloramphenicol-scFv antibody gene and application thereof |
| CN110467677B (en) * | 2019-08-20 | 2022-08-30 | 江苏省农业科学院 | Broad-spectrum recognition single-chain antibody of pyrethroid pesticide metabolite and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1789283A (en) * | 2005-12-13 | 2006-06-21 | 浙江大学 | Artificial antigen, antibody of fenvalerate and uses thereof |
| CN102393465A (en) * | 2011-11-28 | 2012-03-28 | 厦门出入境检验检疫局检验检疫技术中心 | Pyrethroid pesticide monoclonal antibody specificity identification method and application thereof |
-
2013
- 2013-01-25 CN CN201310029812.4A patent/CN103088033B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1789283A (en) * | 2005-12-13 | 2006-06-21 | 浙江大学 | Artificial antigen, antibody of fenvalerate and uses thereof |
| CN102393465A (en) * | 2011-11-28 | 2012-03-28 | 厦门出入境检验检疫局检验检疫技术中心 | Pyrethroid pesticide monoclonal antibody specificity identification method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 农产品中氰戊菊酯农药残留免疫检测技术研究;刘景坤;《中国优秀硕士学位论文全文数据库》;20121015(第10期);6-9、20-32 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103088033A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103861569B (en) | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column | |
| Foord et al. | Production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein 3ABC | |
| CN110526968B (en) | Staphylococcus aureus enterotoxin B nano antibody B7, application and kit | |
| CN110526966B (en) | Staphylococcus aureus enterotoxin B nano antibody B6, application and kit | |
| CN110563839A (en) | Staphylococcus aureus enterotoxin B nano antibody B1, application and kit | |
| CN103088033B (en) | The antibody gene of anti-pyrethroid pesticide scFv and application | |
| CN110577594B (en) | Staphylococcus aureus enterotoxin A nano antibody A21, application and kit | |
| CN113527476A (en) | Novel nano antibody for resisting H5 subtype avian influenza virus and application thereof | |
| CN112457397A (en) | Nano antibody of PRRSV N protein and preparation method and application thereof | |
| CN102936598A (en) | Coding gene of anti-Cry1Ac toxin single-chain variable fragments (scFv) and immuno-polymerase chain reaction (PCR) detection method | |
| CN103361741B (en) | Phage antibody library and application thereof in content determination of clenbuterol hydrochloride | |
| CN107056946A (en) | A kind of Fab antibody and preparation method and application of anti-diethoxy thiophosphate organophosphorus pesticide | |
| CN114213532B (en) | Preparation and application of high-affinity anti-chicken infectious bursal disease virus scFv antibody | |
| Xu et al. | High throughput screening of recombinant antibodies against infectious hematopoietic necrosis virus from a combinatorial antibody library | |
| CN105524170A (en) | Nanobody capable of specifically binding with PSMA (prostate specific membrane antigen) | |
| CN110343161B (en) | Binding protein composition for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH, and preparation method and application thereof | |
| CN103159853A (en) | Completely humanized genetic engineering fragment antigen binding (Fab) antibody against hepatitis C virus core antigen, preparation method thereof and application | |
| CN105177016A (en) | Anti-chloramphenicol-scFv antibody gene and application thereof | |
| CN103288960B (en) | Gene engineering antibody for kitasamycin residue detection, and preparation method and application thereof | |
| CN105085683A (en) | Swine-derived reshaped antibody with insecticidal activity, and preparation method and application thereof | |
| CN103421742B (en) | Recombinant monoclonal antibodies H2 resisting fumonisins B1 | |
| CN104788543B (en) | A kind of zearalenone antibody analog and its application based on polypeptide | |
| CN105319372B (en) | Ractopamine Test paper card and test kit | |
| CN104774267A (en) | Anti-GUS protein monoclonal antibody and applications thereof | |
| CN114702592B (en) | Nanobody for recognizing quetiapine pesticide and enzyme-linked immunoassay method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220712 Address after: 300450 No. 202, building 5, huatangruicheng District 5, Binhai Zhongguancun Science Park, economic and Technological Development Zone, Binhai New Area, Tianjin Patentee after: Tianjin Mingda Baicheng Technology Co.,Ltd. Address before: 300457 No. 29, Thirteenth Avenue, Binhai New Area Economic and Technological Development Zone, Tianjin Patentee before: TIANJIN University OF SCIENCE AND TECHNOLOGY |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150916 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |