CN110467677A - The wide spectrum identification single-chain antibody of pyrethroid pesticide metabolin and its application - Google Patents

The wide spectrum identification single-chain antibody of pyrethroid pesticide metabolin and its application Download PDF

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CN110467677A
CN110467677A CN201910770828.8A CN201910770828A CN110467677A CN 110467677 A CN110467677 A CN 110467677A CN 201910770828 A CN201910770828 A CN 201910770828A CN 110467677 A CN110467677 A CN 110467677A
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chain antibody
phenoxy
metabolin
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thr
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CN110467677B (en
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刘媛
张霄
徐重新
胡晓丹
林曼曼
谢雅晶
张存政
刘贤金
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Jiangsu Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides
    • G01N2430/12—Pyrethroids
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The present invention relates to a kind of Broadspectrum specificity of pyrethroid pesticide metabolin identification single-chain antibody and its applications, the single-chain antibody gene is using one plant of anti-3- phenoxy benzoic acid hybridoma cell strain as template, its heavy chain is expanded respectively and light-chain variable region gene is constructed, and solubility expression is carried out in Escherichia coli, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO.1, and nucleotide sequence is as shown in SEQ ID NO.4；Metabolism/catabolite of the single-chain antibody to a variety of pyrethroid pesticides, i.e. 3- phenoxy benzoic acid, 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol have wide spectrum recognition capability, it has a good application prospect in pyrethroid pesticide metabolin wide spectrum screening detection, the rapid screening detection of single metabolin can also be used for.

Description

The wide spectrum identification single-chain antibody of pyrethroid pesticide metabolin and its application
Technical field
The present invention relates to genetic engineering antibody, especially a kind of pair of pyrethroid pesticide metabolins to know with wide spectrum The single-chain antibody of other ability and its application.
Background technique
Pyrethroid pesticide has many advantages, such as that mammal low toxicity, insecticidal activity are good, is worldwide answered by general (Tang, W etl, Pyrethroid pesticide residues in the global is controlled for agricultural and sanitary insect pest environment:An overview.Chemosphere 2018,191,990-1007.).When pyrethroid pesticide into After entering environment or entering organism by food chain, pyrethroid pesticide is occurred by various biologies or abiotic mode Degradation or metabolism.Wherein this formic acid of 3- phenoxy group (3-phenoxybenzoic acid, 3-PBA), 3- phenoxy benzaldehyde (3- Phenoxybenzaldehyde, PBAld) and 3- phenoxy benzenemethanol (3-phenoxybenzyl alcohol, PBAlc) be Main degradation/the metabolite of pyrethroid pesticide can be used as pyrethrin pesticide biological marker analyte detection (Yusa, V.； Millet,M.；Coscolla,C.；Pardo,O.；Roca,M., Occurrence of biomarkers of pesticide exposure in non-invasive human specimens. Chemosphere 2015,139,91-108.).In addition These three compounds itself also have hormone/antihormonal activity and aquatic toxicity, make to the mankind, biology and Environmental Health At threat (Tyler, C.R.；Beresford,N.； van der Woning,M.；Sumpter,J.P.；Thorpe,K., Metabolism and environmental degradation of pyrethroid insecticides produce compounds with endocrine activities. Environmental Toxicology and Chemistry 2000,19(4),801-809.)。
Pyrethroid pesticide metabolin (3- phenoxy benzoic acid, 3- phenoxy benzaldehyde and 3- phenoxy group benzene at present Methanol) detection method mainly include liquid chromatography and gas chromatography, detection sensitivity can achieve ppb grades of (Abu- Qare,A.W.；Abou-Donia,M.B.,Simultaneous determination of chlorpyrifos, permethrin,and their metabolites in rat plasma and urine by high-performance liquid chromatography.Journal of Analytical Toxicology 2001,25 (4),275-279； Lin,C.H.；Yan,C.T.；Kumar,P.V.；Li,H.P.；Jen,J.F.,Determination of pyrethroid metabolites in human urine using liquid phase microextraction coupled in- syringe derivatization followed by gas chromatography/electron capture detection. Analytical and Bioanalytical Chemistry 2011,401(3),927-937； Mortimer,R.D.； Shields,J.B.,Determination of total free and glucose- conjugated 3-phenoxybenzyl alcohol residues in foods by gas chromatography with electron capture detection. Journal of Aoac International 1996,79(4), 967-971；Bernal,J.L.；Jimenez,J.J.；del Nozal,M.J.；Higes,M.；Llorente,J.,Gas chromatographic determination of acrinathrine and 3-phenoxybenzaldehyde residues in honey.J Chromatogr A 2000, 882(1-2),239-43.).Although these methods are compared with subject to Really, but higher to the degree of dependence of large-scale instrument, pre-treating method is complex, complex steps, and testing cost is higher.
Immunoassay has many advantages, such as that testing cost is low, and big detection flux is the effective substitute technology of chromatography, especially Rapid screening suitable for great amount of samples detects.At present in the world about 3- phenoxy benzoic acid immunoassay method Report, has been respectively adopted polyclonal antibody (Shan, G.；Huang,H.；Stoutamire,D.W.； Gee,S.J.；Leng, G.；Hammock,B.D.,A sensitive class specific immunoassay for the detection of Pyrethroid metabolites in human urine.Chem Res Toxicol 2004,17 (2), 218-25.), Single domain heavy chain antibody (Kim, H.J.；McCoy,M.R.；Majkova,Z.；Dechant,J.E.；Gee, S.J.；Tabares-da Rosa,S.；Gonzalez-Sapienza,G.G.；Hammock,B.D.,Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive Immunoassay.Anal Chem 2012,84 (2), 1165-71.) and anti-3- phenoxy benzoic acid and more anti-immunity compounds Small peptide as biological identification molecule (Gonzalez-Techera, A.；Kim,H.J.；Gee,S. J.；Last,J.A.； Hammock,B.D.；Gonzalez-Sapienza,G.,Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries.Anal Chem 2007,79(23),9191-6.).The although spirit with higher of these antibody Sensitivity, but its specificity is stronger, to the other two kinds of metabolite 3- phenoxy benzaldehydes and 3- phenoxy group benzene of pyrethrin pesticide Methanol does not have wide spectrum recognition reaction, therefore these antibody cannot be used for the wide spectrum detection of both pyrethrin pesticide metabolins.Furthermore Although there is scholar to report the Antibody preparation of identification 3- phenoxy benzenemethanol and glycine compound, author is not provided The antibody to the identification data of the 3- phenoxy benzenemethanol of free state (] Kim, H.J.；Ahn,K.C.；Ma,S.J.；Gee, S.J.；Hammock,B.D.,Development of sensitive immunoassays for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol,a putative human urinary Biomarker for pyrethroid exposure.J Agric Food Chem 2007,55 (10), 3750-7.), therefore not It can determine that the antibody to the compatibility of 3- phenoxy benzenemethanol.It is showed no both at home and abroad to pyrethroid pesticide three classes at present The report of the antibody of metabolin broad spectrum activity identification.
Summary of the invention
In view of the above-mentioned problems, the present invention relates to a kind of energy wide spectrums to identify three kinds of pyrethroid pesticide metabolin (i.e. 3- Phenoxy benzoic acid, 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol) single-chain antibody, and its in these three compounds Application in screening detection.
Present invention firstly provides a kind of wide spectrums of pyrethroid pesticide metabolin to identify single-chain antibody, this is single-stranded Antibody is made of 280 amino acid, and amino acid sequence is for example shown in SEQ ID NO.1.The application is with one plant of identification 3- benzene oxygen The hybridoma of yl benzoic acid be template (the hybridoma preparation method is referring to document: Yuan Liu, Aihua Wu, Jing Hu,Manman Lin,Mengtang Wen,Xiao Zhang,Chongxin Xu,Xiaodan Hu,Jianfeng Zhong,Lingxia Jiao,Yajing Xie,Cunzhen Zhang,Xiangyang Yu, Ying Liang,Xianjin Liu.Detection of 3-phenoxybenzoic acid in river water with a colloidal gold- Based lateral flow immunoassay.Analytical Biochemistry, 2015,483,7-11.), extract Its total serum IgE, reverse transcription cDNA amplify the weight chain variable of mouse using the degenerate primer of mouse antibodies respectively with round pcr Area and light-chain variable region gene.Again by SOE-PCR method, the splicing of single-chain antibody is completed, gene sequencing is eventually passed through and derives Single-chain antibody that its amino acid sequence obtains (amino acid sequence of the single-chain antibody as shown in SEQ ID NO.1, nucleotides sequence Column are as shown in SEQ ID NO.4).The heavy chain variable region of the single-chain antibody forms (its sequence such as SEQ ID by 123 amino acid Shown in NO.2), light chain variable region forms (its sequence is as shown in SEQ ID NO.3) by 107 amino acid.
Secondly, the present invention also provides amino acid sequence single-chain antibodies as shown in SEQ ID NO.1 to detect quasi- deinsectization Application in pyrethrin pesticide metabolin.After the expression vector pET26b (+) that the single-chain antibody gene is transferred to, change turns large intestine bar Bacterium BL21 carries out solubility expression.It is proved by enzyme-linked immunosorbent assay (ELISA), the single-chain antibody is to 3- phenoxy group benzene Formic acid, 3- phenoxy benzenemethanol and 3- phenoxy benzaldehyde have good wide spectrum recognition capability, and its detection sensitivity can Meet the detection demand of pyrethroid pesticide metabolin.
Specifically, above-mentioned " application of the single-chain antibody in detection pyrethroid pesticide metabolin " specifically refers to, Detecting step is as follows:
Coating antigen PBA-OVA is diluted to 2 μ g/mL with CBS buffer, ELISA Plate is added in 100 holes μ L/, and 4 DEG C are overnight, PBST board-washing 3 times.200 holes μ L/ be added 2%MPBS, 37 DEG C warm bath 1h, PBST board-washing 3 times.40 times of 50 μ L dilutions are added in every hole Purifying single-chain antibody (PBS dilution) and 50 μ L 0-20 μ g/mL 3- phenoxy benzoic acids, 3- phenoxy benzaldehyde, 3- benzene oxygen The standard items (10% methanol-PBS dissolution) of base benzyl alcohol.37 DEG C warm bath 1h, PBST board-washing 3 times.1:5000 times of PBS is diluted HRP label anti-His monoclonal antibody, 100 holes μ L/ be added ELISA Plate, 37 DEG C warm bath 1h, PBST board-washing 3 times；The substrate that will now match (H of the 10mg/ml TMB and 25 μ L 0.65% of 100 μ L dimethyl sulfoxides dissolution is added in 10mL CPBS buffer to solution2O2) 100 ELISA Plate, 37 DEG C of standings colour developing 15min are added in the hole μ L/.By 2M H2SO4It is quickly added to 50 holes μ L/ into ELISA Plate, under 450nm Read light absorption value.It is mapped with the ELISA module of Graphpad Prism software, and carries out regression analysis, calculate concentration in inhibiting I50, minimum detection limit I10With linear detection range (I20-I80).Suppression of the 3- phenoxy benzoic acid being calculated to single-chain antibody Final concentration (I processed50) it is 0.55 μ g/mL, minimum detection limit I10For 0.05 μ g/mL, linear detection range (I20-I80) in 0.15- Between 2.64 μ g/mL.The I of 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol to single-chain antibody50Respectively 0.59 and 0.63 μ g/mL.Illustrate that 3- phenoxy benzoic acid, 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol within the above range can lead to Cross above method detection.
In the application, term " pyrethroid pesticide metabolin " refers to 3- this formic acid of phenoxy group (3- Phenoxybenzoic acid, 3-PBA), 3- phenoxy benzaldehyde (3-phenoxybenzaldehyde, PBAld) or 3- benzene One of oxygroup benzyl alcohol (3-phenoxybenzyl alcohol, PBAlc) is a variety of.
Compared with prior art, single-chain antibody provided by the present application can be to three kinds of pyrethroid pesticide metabolism/degradations Principal product in approach is carried out while being detected, without antibody is prepared separately for every kind of compound and is detected respectively, greatly Wide spectrum screening efficiency and testing cost are improved greatly.
Detailed description of the invention
Fig. 1 is the structure figures of pyrethroid pesticide wide spectrum identification single-chain antibody gene and expression vector.
Fig. 2 is the SDS-PAGE analysis chart of the single-chain antibody of affinity purification.
Fig. 3 is standard suppression curve of the 3- phenoxy benzoic acid (3-PBA) to single-chain antibody.
Specific embodiment
The invention will be further described with reference to the accompanying drawing.
Reagent involved in embodiment, instrument source:
The conjugate (PBA-OVA) of 3-PBA hybridoma cell strain (hypotype: IgG1 type), 3-PBA and ovalbumin by Jiangsu Province Agriculture Science Institute agricultural product quality and safety and nutrient research are prepared, save.
TRIzol reagent and SuperScriptTMIII first chain synthetic agent box are purchased from U.S. Invitrogen company.
Primer VH1BACK-NcoI, VH1FOR-2, V κ 2BACK, V κ 4FOR-Not I (V κ 4FOR1-NotI, V κ 4FOR2- NotI, V κ 4FOR3-NotI, V κ 4FOR4-NotI equimolar mixing), LINKBACK, LINKFOR and linker template The sequence of pSW2scD1.3 referring to (2018) Liu Yuan etc. report (Liu Yuan, Liu Min, Zhang Xiao, Xu Chongxin, Lin Manman, Hu Xiaodan, The building Jiangsu agriculture of Zhong Jianfeng, Xie Yajing, Luo Chuping, Zhang Cunzheng, Liu Xianjin .Cry2A toxin antiidiotype single-chain antibody library Industry journal, 2018,34 (5): 1174-1181.), the synthesis of commission Nanjing Genscript Biotechnology Co., Ltd..
2 × Taq PCR master mix and 2 × Pfu PCR master mix is purchased from German DBI company and Dongsheng respectively Biological Co., Ltd.
PET26b (+) carrier by Jiangsu Province Agriculture Science Institute agricultural product quality and safety and nutrient research by being saved.
Restriction enzyme NcoI and NotI, T4 ligase are purchased from U.S. NEB company.
DNA gel QIAquick Gel Extraction Kit, PCR product purification kit, 96 hole elisa Plates are purchased from U.S. Corning company.
Transformed competence colibacillus E.coli BL21 (DE3), skimmed milk power, DEPC water are purchased from the limited public affairs of Beijing Suo Laibao science and technology Department.
Yeast extract and peptone is Oxoid product.Glucose, takes off isopropyl-β-D-thiogalactose (IPTG) Rouge milk powder is purchased from Suo Laibao Biotechnology Co., Ltd.
Kanamicina Solfato, which is purchased from, steps base biology.
Nickel ion affinity chromatograph column (His Trap HP) is purchased from GE Health care company.
The anti-His label mouse monoclonal antibody of HRP label is century Biotechnology Co., Ltd purchased from health.
12% pre-prepared colloid, Tris-MOPS protein electrophoresis buffer and Western blot TMB developing solution (ChromoSensorTMOne-Solution TMB Substrate) it is purchased from Nanjing Genscript Biotechnology Co., Ltd..
3,3,5,5- tetramethyl benzidine (TMB) is purchased from Nanjing Olympic Duo Fu Buddhist nun Biotechnology Co., Ltd.
3-PBA is purchased from U.S. Sigma Aldrich；
3- phenoxy benzaldehyde and 3- phenoxy benzonitrile alcohol are purchased from Aladdin Biotechnology Co., Ltd and source leaf biology respectively Science and Technology Ltd..
Effective cypermethrin, Fenpropathrin and fenvalerate standard items are purchased from Dr.Ehrenstorfer company, Germany.
Phenothrin and decis standard items are purchased from U.S. o2si smart solution company.
Other chemical reagent used in following embodiment and organic solvent are that domestic analysis is pure.
PCR instrument (Takara), metal bath (Hangzhou Europe rice instrument MIULAB), small desk refrigerated centrifuge (Eppendoff), nucleic acid electrophoresis apparatus (6 1 DYY-6C type of Beijing), vertical slab electrophoresis groove (Beijing monarch meaning JY-SCZ2+), electrophoresis Instrument power supply (Beijing monarch anticipate JY600E type), Western blot transferring film electrophoresis apparatus (Beijing monarch anticipate JY-ZY5 type), small desk from Scheming (Eppendorff Centrifuge 5424R), ultrasonic cell disruption instrument (Nanjing month rising sun biotechnology YX- 1000H), microplate reader (Thermo Multiskan GO), board-washing machine (Thermo Wellwash), high-pressure sterilizing pot (Sanyo MLS-3750), ultra low temperature freezer (Haier), pure water meter (Millipore Direct-Q 3UV), balance (good level device FA2004), pH meter (Sartorius PB-10), decolorization swinging table (Scilogex SF-O180-E), shaking table (know Chu's instrument in Shanghai ZQZY-70BF), constant incubator (essence is macro).
1 pyrethroid pesticide metabolin wide spectrum of embodiment identifies single-chain antibody gene and its expression vector establishment
(1) synthesis of the first chain of Total RNAs extraction and cDNA: the miscellaneous of the anti-3- phenoxy benzoic acid frozen in 1 liquid nitrogen is taken Tumor cell strain is handed over, after defrosting at room temperature, 1000g is centrifuged 3min, abandons net supernatant.It is extracted referring to TRIzol reagent specification total The total serum IgE of extraction III kit of SuperScriptTM is synthesized the first chain of cDNA by RNA.
(2) splicing of single-chain antibody gene: the amplification of VH, V kappa gene uses the first chain of cDNA for template, respectively with etc. rub Your VH1BACK-NcoI and VH1FOR-2 is primer and equimolar V κ 2BACK and V κ 4FOR-Not I (V κ 4FOR1- The mixing of NotI, V κ 4FOR2-NotI, V κ 4FOR3-NotI, V κ 4FOR4-NotI equimolar) it is primer, in 1 × Pfu PCR It is expanded in master mix system.Then using pSW2scD1.3 gene as template, LINKBACK and LINKFOR are Linker Primer is expanded in 1 × Pfu PCR master mix.
PCR reaction condition is 94 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1min, 30 circulations, 72 DEG C of 10min. The splicing of next step single-chain antibody gene is used for after the recycling of amplified production glue.
After taking equimolar VH, V κ and linker gene to mix, in 1 × Taq PCR master mix system, carry out First step SOE-PCR, PCR reaction condition is 94 DEG C of 1min, 65 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72 degree of 7min.First After walking 1000 times of the sterile water dilution of SOE-PCR product, 1 containing primer VH1BACK-NcoI and V κ 4FOR1-NotI is added × Taq PCR master mix system.PCR condition setting is 94 DEG C of 40s, 55 DEG C of 40s, 72 DEG C of 2min, 5 circulations；94 DEG C, 40s, 50 DEG C of 40s, 72 DEG C of 2min, 5 circulations；94 DEG C of 40s, 45 DEG C of 40s, 72 DEG C of 2min, 5 circulations；94 DEG C of 40s, 40 DEG C 40s, 72 DEG C of 2min, 15 circulations；72℃7min.
(3) expression vector establishment: the SOE-PCR product to 1 μ g glue recovery purifying and 1 μ g pET26b (+) plasmid respectively, Using I double digestion of Not I and Noc.After 37 DEG C of digestions overnight, 80 DEG C of inactivation 10min, digestion products and plasmid, with T4 ligase 16 DEG C connection overnight, 65 DEG C of inactivation 10min, PCR product Purification Kit, removal ligase and buffer in salt from Subconstiuent, then it is spare with sterile water dissolution.It is placed in competence is turned from -80 DEG C of 50 μ L E.coli BL21 (DE3) taken out changes It thaws on ice, 2 μ l purifying connection product (about 100ng) is added, it is soft to mix.30min, 42 DEG C of water-bath thermal shocks are placed on ice 90s is placed in ice face 3min, and 2 × TY of 1mL culture medium, 37 DEG C of 200rpm shake culture 1h are added.It takes 100 μ L to be coated with one piece to contain There are the TYE plate of 1% glucose and 50 μ g/mL kanamycins, 37 DEG C of inversion overnight incubations.Next day picking single colonie, access 2 × TY culture medium containing 1% glucose and 50 μ g/mL kanamycins, 37 DEG C, after 250rpm is incubated overnight, after bacterium solution sequencing, Nucleic acid and amino acid sequence analysis are carried out with 5.5 software of Vector NIT suite.And with small amount plasmid extraction kit, mention Plasmid is taken to carry out double digestion verifying.
Fig. 1 (A) shows the PCR amplification of VH, V κ and linker gene as a result, wherein VH gene, V kappa gene molecular weight exist For 250bp between 500bp, linker gene is located at the position on the lower side 100bp, the compound expection of molecular size range.
Fig. 1 (B) shows the splicing result to SOE-PCR, there is a clear band near 800bp, and clip size meets The molecular weight of single-chain antibody is expected, and single-chain antibody gene constructs successfully.
After single-chain antibody gene is connected to pET26b (+) carrier, change turns competence BL21 (DE3), and random picking 10 Clone, extract plasmid after with NcoI and Not I double digestion verify, wherein 3 clone release purpose band be located at 500bp with Between 1000bp (Fig. 1 C), the expression vector establishment success of single-chain antibody gene, the amino acid sequence of the single-chain antibody such as SEQ Shown in ID NO.1.
In specific implementation, can also the nucleotide sequence according to disclosed in SEQ ID NO.4, the artificial synthesized simply connected is anti- Body.
2 pyrethroid pesticide metabolin wide spectrum of embodiment identifies the sequence analysis of single-chain antibody
Utilize the on-line analysis tool (www.expasy.org/tools/ of Bioinformatics Resource portal ExPASy Protparam.html), the amino acid sequence for inputting single-chain antibody, for calculate and predict single-chain antibody amino acid quantity, The physicochemical properties such as molecular formula, molecular weight, theoretical isoelectric point, stability.Utilize international immune genetic information system IMGT database Online tool (http://www.imgt.org/IMGT_vquest/vquest), be set as species be mouse, type is immune Globulin (IG) and single-chain antibody option, submit anti-single-chain antibody nucleic acid sequence, and analysis single-chain antibody weight light chain variable region is compiled The code affiliated allele of genetic fragment, the information such as framework region and CDR region mark.
Test utilize ExPASy on-line analysis tool, submit single-chain antibody nucleic acid sequence, be calculated single-chain antibody by 280 amino acid composition, molecular formula C1327H2040N366O404S10, molecular weight be 29905.61 (molecular size range with Fig. 3 result is consistent), theoretical isoelectric point is 8.95.Negative charge amino acid (Asp+Glu) total number of residues is 17, positive charge ammonia Base acid (Arg+Lys) total number of residues is 22.It is 30h that the antibody is theoretical in the granulophilocyte of mammal, in yeast cells It is interior to be greater than 20h, 10h is greater than in Escherichia coli.Instability index is 55.58, is incorporated into labile protein.Aliphatic amines Sour coefficient is 69.39, hydrophily average index -0.239, is hydrophilic protein.
By single-chain antibody nucleic acid sequence be committed to it is after IMGT database analysis shows.The VH 369bp at 5 ' ends, 3 ' ends V κ length is 321bp.VH with V κ is connected by the linker sequence of 45bp.The His label of 18 bp is also had in end.VH by V, D, J genetic fragment form, wherein with the highest allele of V genetic fragment consistency be Musmus IGHV1-18*01F or Musmus IGHV1-22*01F, consistency 89.93%.It is Musmus with the highest allele of J genetic fragment consistency IGHJ1*01F or Musmus IGHJ1*03F (consistency=90.57%), with the highest allele of D genetic fragment consistency It is Musmus IGHD1-1*01 F.4 Framework Region amino acid length of IMGT mark VH are respectively 25-17-38-11, and the 3 of VH A area CDR amino acid length is respectively 8-8-16.
V κ is encoded by V, J genetic fragment, is Musmus IGKV4-72* respectively with the highest allele of the two consistency 01F (consistency=94.57%) and Musmus IGKJ1*01F (consistency=100%).4 framework regions of V κ of IMGT mark Three CDR region amino acid lengths of amino acid length 26-17-36-10, V κ are 5-3-9.
3. pyrethroid pesticide metabolin wide spectrum of embodiment identifies the solubility expression experiment of single-chain antibody
Specific step is as follows for the present embodiment:
Amino acid sequence single-chain antibody as shown in SEQ ID NO.1 prepared by the embodiment 1 for taking 50 μ L-80 DEG C to freeze Strain glycerol stocks are added 2 × TY of 5mL (containing 1% glucose and 50 μ g/mL kanamycins), 37 DEG C, 250rpm shake culture Overnight.By 2.5mL overnight culture, 2 × TY of 250mL (glucose and 50 μ g/mL cards containing final concentration 0.1% is added in next day That mycin) 1L triangular flask, 37 DEG C, 250rpm shake culture to OD600=0.9 (about 3h).It is added final concentration 0.25mM's IPTG, 16 DEG C, 250rpm shake culture cultivates 8h.4 DEG C of culture solution, 1800g is centrifuged 10min, abandons supernatant.Remaining thallus is used 125mL PBS is resuspended, and mediates and is uniformly mixed, is placed in ultrasonication in ice-water bath.Ultrasound condition are as follows: output power 30%, 25 Degree, mode 06, working time 30min, ultrasonic 3s are spaced 4s.4 DEG C, 10000g, it is centrifuged after 30min that take supernatant to obtain full bacterium broken Broken supernatant (S2).Purified with the single-chain antibody in HisTrap HP column S2, purification process is referring to GE Products explanation Book, the single-chain antibody obtained after purification are analyzed for SDS-PAGE, in addition with Bradford method measurement purifying protein concentration (ginseng See document: " burgess RR, her more thorough MP. protein purification guide (the former book second edition) Beijing [M]: Science Press； 2013,69-70 ").
Testing result is as shown in Fig. 2, in Fig. 2, and swimming lane 1: sample before upper prop, swimming lane 2: upper prop is pierced by liquid, swimming lane M: albumen Molecular weight Marker, swimming lane 3:50mM imidazoles elution fraction, swimming lane 4:100mM imidazoles elution fraction, swimming lane 5:200mM imidazoles Elution fraction.As it can be seen that test obtains electrophoretically pure single-chain antibody band under the imidazoles elution requirement of 200mM, by albumen The yield of concentration calculation single-chain antibody is 2.68 mg/L.
4 pyrethroid pesticide metabolin wide spectrum of embodiment identifies single-chain antibody to 3- phenoxy benzoic acid, 3- benzene oxygen The detection application of benzaldehyde and 3- phenoxy benzenemethanol
This test establishes 3- phenoxy benzoic acid (3- with the single-chain antibody after purification that embodiment 3 obtains respectively PBA), the competitive ELISA detection method of 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol, the specific steps are as follows:
Coating antigen PBA-OVA is diluted to 2 μ g/mL with CBS buffer, ELISA Plate is added in 100 holes μ L/, and 4 DEG C are overnight, PBST board-washing 3 times.200 holes μ L/ be added 2%MPBS, 37 DEG C warm bath 1h, PBST board-washing 3 times.40 times of 50 μ L dilutions are added in every hole Purifying single-chain antibody (PBS dilution) and 50 μ L 0-20 μ g/mL 3- phenoxy benzoic acids, 3- phenoxy benzaldehyde or 3- benzene Oxygroup benzyl alcohol standard items (10% methanol-PBS dissolution).37 DEG C warm bath 1h, PBST board-washing 3 times.1:5000 times of PBS is diluted HRP label anti-His monoclonal antibody, 100 holes μ L/ be added ELISA Plate, 37 DEG C warm bath 1h, PBST board-washing 3 times.The substrate that will now match (H of the 10mg/ml TMB and 25 μ L 0.65% of 100 μ L dimethyl sulfoxides dissolution is added in 10mL CPBS buffer to solution2O2) 100 ELISA Plate, 37 DEG C of standings colour developing 15min are added in the hole μ L/.By 2M H2SO4It is quickly added to 50 holes μ L/ into ELISA Plate, under 450nm Read light absorption value.
With the log concentration of 3- phenoxy benzoic acid, 3- phenoxy benzaldehyde or 3- phenoxy benzenemethanol respectively to corresponding B/B0It is worth (ratio that standard items correspond to light absorption value with light absorption value corresponding when zero standard's product) mapping drafting standard and inhibits bent Line carries out regression analysis with the ELISA module of Graphpad Prism software, calculates concentration I in inhibiting50, minimum detection limit I10With linear detection range (I20-I80)。
Above-mentioned standard curve method for building up be this field routine techniques, as document " Liu Yuan, Zhang Cun-zhen, Yu Xiang-yang,Zhang Zhi-yong,Zhang Xiao,Liu Rong-rong,Liu Xian-jin, Gong Zhen-ming.Establishment and evaluation of immunoassay for zeranol in bovine Urine.Journal of Zhejiang University Science is B.2007,8 (12): the side of the reports such as 900-905. " Method.
Testing result is as shown in figure 3, in Fig. 3, B/B0It represents 3-PBA standard items and corresponds to light absorption value and zero standard's product When correspond to the ratio of light absorption value.By the regression analysis of standard curve, 3- phenoxy benzoic acid is calculated to single-chain antibody Inhibit final concentration (I50) it is 0.55 μ g/mL, minimum detection limit I10For 0.05 μ g/mL, linear detection range (I20-I80) In Between 0.15-2.64 μ g/mL.In addition the I to single-chain antibody of 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol50Respectively 0.59 and 0.63 μ g/mL, single-chain antibody have 3- benzene oxygen formic acid, 3- phenoxy benzaldehyde and 3- phenoxy benzenemethanol similar Recognition capability, have Broadspectrum specificity.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>the wide spectrum identification single-chain antibody of pyrethroid pesticide metabolin and its application
<141> 2019-08-20
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 280
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Ala Gln Val Gln Leu Gln Gln Ser Gly
20 25 30
Pro Glu Leu Val Gln Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr
35 40 45
Ser Gly Tyr Thr Phe Thr Glu Phe Thr Met His Trp Val Lys Gln Ser
50 55 60
His Gly Lys Ser Leu Glu Trp Ile Gly Gly Ile Asn Pro Ala Asn Gly
65 70 75 80
Glu Thr Arg Leu Asn Gln Lys Phe Lys Val Arg Ala Thr Leu Thr Val
85 90 95
Asp Thr Ser Ser Ser Thr Ala Tyr Met Asp Leu Arg Ser Leu Thr Ser
100 105 110
Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Val Tyr Tyr Tyr Gly Gly
115 120 125
Gly Leu Arg Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr
130 135 140
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser
165 170 175
Pro Gly Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser
180 185 190
Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp
195 200 205
Ile Tyr Ala Thr Ser Ile Leu Ala Ser Gly Val Pro Ala Arg Phe Thr
210 215 220
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Leu Ser Arg Val Glu
225 230 235 240
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Leu Trp Arg Ile Asn Pro
245 250 255
Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala
260 265 270
Leu Glu His His His His His His
275 280
<210> 2
<211> 123
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Gln Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Ala Asn Gly Glu Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Tyr Tyr Tyr Gly Gly Gly Leu Arg Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 3
<211> 107
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Ile Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Leu Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Leu Trp Arg Ile Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 4
<211> 840
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccatgg cccaggtcca gctgcagcag tctgggcctg agctggtgca gcctggggct 120
tcagtgaaga tatcctgcaa gacctctgga tacacattca ctgaattcac catgcactgg 180
gtgaagcaga gccacggaaa gagccttgag tggattggag gtattaatcc tgcaaatggt 240
gaaactaggc tcaaccagaa gttcaaggtc agggccacat tgactgtgga cacgtcctcc 300
agcacagcct acatggacct ccgcagcctg acatctgagg attctgcagt ctattactgt 360
gcacgagtgt attactacgg tggtggcctg cgctggtact tcgatgtctg gggccaaggg 420
accacggtca ccgtctcctc aggtggaggc ggttcaggcg gaggtggcag cggcggtggc 480
gggtcggaca ttgagctcac ccagtctcca gcaatcctgt ctgcatctcc aggggagagg 540
gtcacaataa cttgcagggc cagctcaagt gtaagttaca tgcactggta ccagcagaag 600
ccaggatcct cccccaaacc ctggatttat gccacttcca tcctggcttc tggagtccct 660
gctcgcttca ctggcagtgg gtctgggact tcttactctc tcacactcag cagagtggag 720
gctgaagatg ctgccactta ttactgccag ctgtggagaa ttaacccacc gacgttcggt 780
ggaggcacca agctggaaat caaacgggcg gccgcactcg agcaccacca ccaccaccac 840

Claims (4)

1. a kind of wide spectrum of pyrethroid pesticide metabolin identifies single-chain antibody, which is characterized in that the single-chain antibody Amino acid sequence is as shown in SEQ ID NO.1.
2. single-chain antibody as described in claim 1, which is characterized in that the single-chain antibody includes amino acid sequence such as SEQ ID The light chain variable region as shown in SEQ ID NO.3 of heavy chain variable region and amino acid sequence shown in NO.2.
3. single-chain antibody as described in claim 1, which is characterized in that the nucleotide sequence of the single-chain antibody such as SEQ ID Shown in NO.4.
4. application of the single-chain antibody a method according to any one of claims 1-3 in pyrethroid pesticide metabolism analyte detection.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103088033A (en) * 2013-01-25 2013-05-08 天津科技大学 Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene
CN108101970A (en) * 2017-12-14 2018-06-01 江苏省农业科学院 Based on the Cry1Ab toxin analogue antigen of antiidiotype nano antibody and its application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103088033A (en) * 2013-01-25 2013-05-08 天津科技大学 Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene
CN108101970A (en) * 2017-12-14 2018-06-01 江苏省农业科学院 Based on the Cry1Ab toxin analogue antigen of antiidiotype nano antibody and its application

Non-Patent Citations (1)

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Title
吴元元等: "拟除虫菊酯类农药单链可变区抗体的制备、鉴定及其特异性分析", 《食品安全质量检测学报》 *

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