CN110467677B

CN110467677B - Broad-spectrum recognition single-chain antibody of pyrethroid pesticide metabolite and application thereof

Info

Publication number: CN110467677B
Application number: CN201910770828.8A
Authority: CN
Inventors: 刘媛; 张霄; 徐重新; 胡晓丹; 林曼曼; 谢雅晶; 张存政; 刘贤金
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2022-08-30
Anticipated expiration: 2039-08-20
Also published as: CN110467677A

Abstract

The invention relates to a broad-spectrum specificity recognition single-chain antibody of pyrethroid pesticide metabolite and application thereof, wherein the single-chain antibody gene is constructed by respectively amplifying heavy chain and light chain variable region genes of an anti-3-phenoxybenzoic acid hybridoma cell strain serving as a template and is subjected to soluble expression in escherichia coli, the amino acid sequence of the single-chain antibody is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 4; the single-chain antibody has broad-spectrum recognition capability on various metabolism/degradation products of pyrethroid pesticides, namely 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol, has good application prospect in broad-spectrum screening detection of pyrethroid pesticide metabolites, and can also be used for rapid screening detection of single metabolites.

Description

Broad-spectrum recognition single-chain antibody of pyrethroid pesticide metabolite and application thereof

Technical Field

The invention relates to a genetic engineering antibody, in particular to a single-chain antibody with broad-spectrum recognition capability on pyrethroid pesticide metabolites and application thereof.

Background

Pyrethroid pesticides have the advantages of low mammalian toxicity, good insecticidal activity and the like, and are widely applied to agricultural and sanitary pest control worldwide (Tang, W etl, Pyrethoid pesticide residues in the viral environment: An overview. Chemospere 2018,191, 990-. Pyrethroid pesticides degrade or metabolize by a variety of biological or non-biological means after entering the environment or through the food chain into the organism. Wherein 3-phenoxy basic formic acid (3-phenoxy acid,3-PBA), 3-phenoxy benzaldehyde (3-phenoxy benzaldehyde, PBAll) and 3-phenoxy benzyl alcohol (3-phenoxy benzyl alcohol, PBAllc) are main degradation/metabolic products of the pyrethroid pesticide, and can be used as a pyrethroid pesticide biomarker for detection (Yusa, V.; Millet, M.; Coscolla, C.; Pardo, O.; Roca, M., Occurence of biomarkers of pesticide in non-innovative human spectra 2015,139, 91-108.). These three compounds also have hormonal/anti-hormonal activity and aquatic toxicity themselves, posing a threat to human, biological and environmental health (Tyler, c.r.; Beresford, n.; van der Woning, m.; summer, j.p.; Thorpe, k., metabolic and environmental degradation of biochemical products with end activities, environmental Toxicology and Chemistry 2000,19(4), 801-.

The detection methods of the current pyrethroid pesticide metabolites (3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol) mainly comprise liquid chromatography and gas chromatography, and the detection sensitivity can reach ppb level (Abu-Qare, A.W.; Abou-Donia, M.B., Simultaneous detection of chlorodiferos, permethrin, and the microorganisms in a plate and a urea by high-purity chromatography. journal of Analytical nomenclature 2001,25(4), 275-279; Lin, C.H.; Yan C.T.; Kumar, P.V.; Li, DeH.P.; Jen, J.F., detection of pyrethroid, biochemical analysis, and 3-phenoxy chromatography, and 5. D.7. conversion, molecular analysis, and detection methods of biochemical analysis, and 3-biochemical analysis, and 7. J.F., hydrolysis, fluorescence analysis, fluorescence detection, journal of aoac International 1996,79(4), 967-; bernal, j.l.; jimenez, j.j.; del Nozal, m.j.; higes, m.; llorente, J., Gas chromatography determination of acrinathrine and 3-phenoxybenzadheide reactions in honey. J.chromamer A2000,882 (1-2), 239-43.). Although the methods are accurate, the dependence on large instruments is high, the pretreatment method is complex, the steps are complicated, and the detection cost is high.

The immunoassay method has the advantages of low detection cost, large detection flux and the like, is an effective alternative technology of chromatography, and is particularly suitable for rapid screening detection of a large number of samples. There have been reports of immunoassays for 3-phenoxybenzoic acids internationally, using polyclonal antibodies (Shan, G.; Huang, H.; Stoutamide, D.W.; Gee, S.J.; Leng, G.; Hammark, B.D., A sensitive class specific immunological assay for the detection of refractory metals in human urine. chem.Res. Toxicol 2004,17(2),218-25.), heavy chain single domain antibodies (Kim, H.J.; McCoy, M.R.; Majkova, Z.; Decant, J.E.; Gee, S.J.; Tabares-da Rosa, S.E.; Gonlez e-pienza, G.G.; B.D., Isalel.; Gee.J.; S.J.; Tabares-da Rosa, S.S.Saybone, S.E., Gonza-piez.; G.G., B.D., Isalel., Isales.J.; Gee., S.J.; E.J.; E.E.J.; Gee.J.; E.E.J. Sambose., S.J.; C.S.J. Thomuch C.S.S.J.; E.S.A.A. Tn.A. A. and C.A. A. Biotin. immunological complexes, and C.; anti-Chem. chem.84, and Goodynamic.; A. 5, and Goodyne, 7. Chem.; antibodies, 7. immunological complexes, J.A.; hammock, b.d.; Gonzalez-Sapienza, G., polymeric antibody-based mutant immunological assay for small analytes with short peptide loops isolated from phase libraries 2007,79(23), 9191-6.). Although the antibodies have high sensitivity, the antibodies have strong specificity, and do not have broad-spectrum recognition effect on other two metabolites of the pyrethroid pesticide, namely 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol, so the antibodies cannot be used for broad-spectrum detection of the two metabolites of the pyrethroid pesticide. Furthermore, although the preparation of antibodies recognizing the complex of3-phenoxybenzyl alcohol and glycine was reported by the scholars, the authors did not give the data on the recognition of the free 3-phenoxybenzyl alcohol (] Kim, H.J.; Ahn, K.C.; Ma, S.J.; Gee, S.J.; Hammick, B.D., Development of sensitive immunological analysis for the detection of the hydrolysis joint of 3-phenyloxybenzalkyl alcohol, a pure human amino biology chemistry syndrome for hydrolysis reaction.J. agricultural Food m 2007,55(10),3750-7.), so the affinity of the antibody for 3-phenoxybenzyl alcohol could not be determined. At present, no report on an antibody for identifying three metabolites of the pyrethroid pesticide in a broad spectrum is seen at home and abroad.

Disclosure of Invention

Aiming at the problems, the invention relates to a single-chain antibody capable of identifying three pyrethroid pesticide metabolites (namely 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol) in a broad spectrum manner and application thereof in screening and detecting the three compounds.

The invention firstly provides a broad-spectrum recognition single-chain antibody of pyrethroid pesticide metabolites, which consists of 280 amino acids, and the amino acid sequence of the single-chain antibody is shown as SEQ ID NO. 1. A hybridoma recognizing 3-phenoxybenzoic acid is used as a template (the preparation method of the hybridoma is shown in the literature: Yuan Liu, Aihua Wu, Jung Hu, Man Lin, Mengtang Wen, Xiao Zhuang, Chongxin Xu, Xiaodan Hu, Jianfeng Zhong, Lingxia Jian, Yaying Xie, Cunzhen Zhang, Xiangyang Yu, Ying Liung Huang, Xiajin Liu, detection of3-phenoxy benzoic acid in river water with a colloidal gold-based molecular amplification experiment. And then completing the splicing of the single-chain antibody by an SOE-PCR method, and finally performing gene sequencing and deducing the amino acid sequence of the single-chain antibody to obtain the single-chain antibody (the amino acid sequence of the single-chain antibody is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 4). The heavy chain variable region of the single-chain antibody consists of 123 amino acids (the sequence of which is shown in SEQ ID NO. 2), and the light chain variable region consists of 107 amino acids (the sequence of which is shown in SEQ ID NO. 3).

Secondly, the invention also provides application of the single-chain antibody with the amino acid sequence shown as SEQ ID NO.1 in detection of pyrethroid pesticide metabolites. After the single-chain antibody gene is transferred into an expression vector pET26b (+), the single-chain antibody gene is transformed into escherichia coli BL21 for soluble expression. Enzyme-linked immunosorbent assay (ELISA) proves that the single-chain antibody has good broad-spectrum recognition capability on 3-phenoxybenzoic acid, 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, and the detection sensitivity can meet the detection requirement of pyrethroid pesticide metabolites.

Specifically, the application of the single-chain antibody in detection of pyrethroid pesticide metabolites specifically means that the detection steps are as follows:

the original PBA-OVA coating was diluted to 2. mu.g/mL with CBS buffer, 100. mu.L/well was added to the microplate, overnight at 4 ℃ and the plate was washed 3 times with PBST. Add 2% MPBS to 200. mu.L/well, incubate at 37 ℃ for 1h, wash plate 3 times with PBST. 50 μ L of purified single chain antibody (PBS diluted) diluted 40 fold and 50 μ L of standards of 0-20 μ g/mL 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde, 3-phenoxybenzyl alcohol (10% methanol-PBS solubilized) were added to each well. The plate was washed 3 times with PBST after 1h incubation at 37 ℃. Adding HRP-labeled anti-His monoclonal antibody diluted by 1:5000 times PBS into an ELISA plate at a concentration of 100 mu L/hole, carrying out warm bath at 37 ℃ for 1h, and washing the plate for3 times by PBST; the substrate solution was freshly prepared (10mL of CPBS buffer was added to 100. mu.L of 10mg/mL TMB dissolved in dimethylsulfoxide and 25. mu.L of 0.65% H ₂ O ₂ ) Adding 100 mu L/hole enzyme label plate, standing at 37 ℃ for color development for 15 min. 2M H ₂ SO ₄ Add 50. mu.L/well quickly to the microplate and read the absorbance at 450 nm. The ELISA module of Graphpad Prism software was used for mapping and regression analysis to calculate the concentration I in inhibition ₅₀ Minimum detection Limit I ₁₀ And linear detection range (I) ₂₀ -I ₈₀ ). Calculating the final inhibitory concentration (I) of the obtained 3-phenoxybenzoic acid to the single-chain antibody ₅₀ ) 0.55 mu g/mL and the lowest detection limit I ₁₀ 0.05. mu.g/mL, linear detection range (I) ₂₀ -I ₈₀ ) Between 0.15 and 2.64. mu.g/mL. 3-Phenoxybenzaldehyde and 3-Phenoxybenzyl alcohol on Single chain antibody I ₅₀ 0.59 and 0.63. mu.g/mL, respectively. It is explained that 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol within the above ranges can be detected by the above method.

In the present application, the term "pyrethroid pesticide metabolite" refers to one or more of 3-phenoxybenzoic acid (3-PBA), 3-phenoxybenzaldehyde (PBAld), or 3-phenoxybenzyl alcohol (3-phenoxybenzyl alcohol, PBAlc).

Compared with the prior art, the single-chain antibody provided by the application can be used for simultaneously detecting main products in three pyrethroid pesticide metabolism/degradation ways, and the antibody is not required to be prepared independently and detected respectively for each compound, so that the broad-spectrum screening efficiency and the detection cost are greatly improved.

Drawings

FIG. 1 is a construction diagram of a pyrethroid pesticide broad-spectrum recognition single-chain antibody gene and an expression vector.

FIG. 2 is an SDS-PAGE analysis of affinity purified single chain antibodies.

FIG. 3 is a standard inhibition curve of 3-phenoxybenzoic acid (3-PBA) against single chain antibodies.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Reagents, instrument sources referred to in the examples:

3-PBA hybridoma cell strain (subtype: IgG1 type), and conjugate (PBA-OVA) of 3-PBA and ovalbumin are prepared and stored by quality safety and nutrition research of agricultural products of academy of agricultural sciences of Jiangsu province.

TRIzol reagent and SuperScript ^TM III first Strand Synthesis kit was purchased from Invitrogen, USA.

The sequences of the primers VH1BACK-NcoI, VH1FOR-2, V κ 2BACK, V κ 4FOR-Not I (molar mixture of V κ 4FOR1-NotI, V κ 4FOR2-NotI, V κ 4FOR3-NotI, V κ 4FOR4-NotI, etc.), LINKBACK, LINKFOR and linker template pSW2scD1.3 are reported in Liu et al (2018) (Liu, Liumin, Zhang Campsis, Xuezui, Mannman, Sudan, Zhongjian Feng, Xiayao, Churoriping, Zhang Zheng, Liujin. Cry2A toxin anti-idiotypic single-chain antibody library construction. Jingsu agriculture report, 2018,34(5): 1174. supplement 1181.), and Seityuri biological Limited Biotech.

2 XTaq PCR master mix and 2 Xpfu PCR master mix were purchased from DBI, Germany, and Dongsheng organisms, Inc., respectively.

The pET26b (+) vector is preserved in agricultural product safety and nutrition research of Jiangsu province academy of agricultural sciences.

Restriction enzymes NcoI and NotI, T4 ligase were purchased from NEB, USA.

DNA gel recovery kit, PCR product purification kit, and 96-well plate were purchased from Corning, USA.

Coli BL21(DE3), skim milk powder, DEPC water were purchased from beijing solibao technologies ltd.

Yeast extract and peptone are Oxoid products. Glucose, isopropyl-beta-D-thiogalactoside (IPTG), and skimmed milk powder were obtained from Solebao Biotechnology Ltd.

Kanamycin monosulfate was purchased from mylar organisms.

Nickel ion affinity chromatography columns (His Trap HP) were purchased from GE Health care.

HRP-labeled anti-His-tag murine monoclonal antibody was purchased from japan biotechnology limited.

12% preformed gel, Tris-MOPS protein electrophoresis buffer solution and Western blot TMB color development solution (chromoSensor) ^TM One-Solution TMB Substrate) was purchased from tsingtaury biotechnology limited, tokyo.

3,3,5, 5-Tetramethylbenzidine (TMB) was purchased from Odofny Biotech, Inc., Nanjing.

3-PBA was purchased from Sigma Aldrich, USA;

3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol were purchased from Aladdin Biotech Ltd and Source leaf Biotech Ltd, respectively.

Cyhalothrin, fenpropathrin and fenvalerate standards were purchased from dr.

Phenothrin and deltamethrin standards were purchased from o2si smart solution, usa.

Other chemical reagents and organic solvents used in the following examples are all domestic analytical purifications.

PCR instrument (Takara), metal bath (Hangzhou Europe rice instrument MIULAB), small-sized table-type refrigerated Centrifuge (Eppendoff), nucleic acid electrophoresis instrument (Beijing Liuyi DYY-6C type), vertical plate electrophoresis tank (Beijing Junyi JY-SCZ2+), electrophoresis instrument power supply (Beijing Junyi JY600E type), Western blot membrane transfer electrophoresis instrument (Beijing Junyi JY-ZY5 type), small-sized table-type Centrifuge (Eppendoff Centrifuge 5424R), ultrasonic cell disruption instrument (Nanjing Yuxu Biotechnology YX-1000H), enzyme labeling instrument (Thermo Multiskan GO), plate washing machine (Thermo Wellwash), autoclave (Sanyo MLS-3750), ultra-low temperature refrigerator (Hal), pure water instrument (Millipore-Q3 UV balance), Liangping instrument FA (Liangping Heng instrument FA), pH meter (Sartorius-10), Sagel shaking table (Scilox-3750), ultra-low temperature refrigerator (Hai Zhao BF-180 instrument), super-BF-1000H, Constant temperature incubator (Jinghong).

Example 1 pyrethroid pesticide metabolite broad-spectrum recognition single-chain antibody gene and expression vector construction thereof

(1) Total RNA extraction and cDNA first strand synthesis: taking 1 hybridoma cell strain resisting 3-phenoxybenzoic acid frozen in liquid nitrogen, thawing, centrifuging for 3min at room temperature at 1000g, and discarding the supernatant. Total RNA was extracted according to the TRIzol reagent instructions, and the extracted total RNA was synthesized into the first strand cDNA using the SuperScriptTM III kit.

(2) Splicing of single-chain antibody genes: amplification of VH and V.kappa.genes was carried out in a 1 Xpfu PCR master mix system using the first strand of cDNA as template, equimolar VH1BACK-NcoI and VH1FOR-2 as primers, and equimolar V.kappa.2 BACK and V.kappa.4 FOR-Not I (V.kappa.4 FOR1-NotI, V.kappa.4 FOR2-NotI, V.kappa.4 FOR3-NotI, V.kappa.4 FOR4-NotI, etc. mixed as primers). Linker was amplified in 1 Xpfu PCR master mix using pSW2scD1.3 gene as template and LINKBACK and LINKFOR as primers.

The PCR reaction conditions are 94 ℃ for 5min, 94 ℃ for 45s, 55 ℃ for 45s, 72 ℃ for 1min, 30 cycles and 72 ℃ for 10 min. And recovering the amplification product glue for the next step of splicing the single-chain antibody gene.

Mixing VH, Vkappa and linker genes with equal molar, and performing SOE-PCR in a 1 xTaq PCR master mix system for the first step, wherein the PCR reaction conditions are 94 ℃ for 1min, 65 ℃ for 1min, 72 ℃ for 1min and 30 cycles; 72 degrees 7 min. The SOE-PCR product of the first step was diluted 1000-fold with sterile water and added to a 1 XTAQ PCR master mix system containing the primers VH1BACK-NcoI and V.kappa.4 FOR 1-NotI. The PCR conditions were set to 94 ℃ for 40s, 55 ℃ for 40s, 72 ℃ for 2min, 5 cycles; 94 ℃,40 s, 50 ℃ and 40s, 72 ℃ for 2min, 5 cycles; 94 ℃ for 40s, 45 ℃ for 40s, 72 ℃ for 2min, 5 cycles; at 94 ℃ for 40s, at 40 ℃ for 40s, at 72 ℃ for 2min, and for 15 cycles; 7min at 72 ℃.

(3) Construction of an expression vector: the purified SOE-PCR product and 1. mu.g of pET26b (+) plasmid were recovered from 1. mu.g of the gel, and digested simultaneously with Not I and Noc I. After enzyme digestion at 37 ℃ overnight, inactivation is carried out at 80 ℃ for 10min, the enzyme digestion products and plasmids are connected by T4 ligase at 16 ℃ overnight and inactivated at 65 ℃ for 10min, a PCR product purification kit is purified, the salt ion components in the ligase and the buffer solution are removed, and the mixture is dissolved by sterile water for standby. 50 μ L of E.coli BL21(DE3) transcompetent from-80 ℃ was thawed on ice, 2 μ L of purified ligation product (ca. 100ng) was added and mixed gently. Placing on ice for 30min, heating in water bath at 42 deg.C for 90s, placing on ice surface for 3min, adding 1mL 2 × TY culture medium, and shake culturing at 37 deg.C 200rpm for1 h. 100 μ L of TYE plate containing 1% glucose and 50 μ g/mL kanamycin was spread and cultured overnight at 37 ℃ in an inverted state. The next day, a single colony was picked, inoculated into 2 XTY medium containing 1% glucose and 50. mu.g/mL kanamycin, incubated overnight at 37 ℃ and 250rpm, and after sequencing of the inoculum, nucleic acid and amino acid sequence analysis was performed using Vector NIT suite 5.5 software. And extracting the plasmid by using a plasmid small quantity extraction kit for double enzyme digestion verification.

FIG. 1(A) shows the PCR amplification results of VH and Vkappa genes, which have molecular weights of 250bp to 500bp, and linker genes, which are located at positions lower than 100bp and have molecular weights as expected.

FIG. 1(B) shows the splicing result of SOE-PCR, a clear band is formed around 800bp, the fragment size is in line with the molecular weight expectation of the single-chain antibody, and the single-chain antibody gene is successfully constructed.

After the single-chain antibody gene is connected to a pET26b (+) vector, transformation competence BL21(DE3) is achieved, 10 clones are randomly picked, plasmids are extracted, and double enzyme digestion verification is carried out by NcoI and Not I, wherein target bands released by 3 clones are located between 500bp and 1000bp (shown in figure 1C), the construction of an expression vector of the single-chain antibody gene is successful, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 1.

In specific implementation, the monoclonal antibody can also be artificially synthesized according to the nucleotide sequence disclosed by SEQ ID NO. 4.

Example 2 sequence analysis of pyrethroid pesticide metabolite broad-spectrum recognition Single-chain antibody

An online analysis tool (www.expasy.org/tools/protparam. html) of a bioinformatics resource portal ExPASy is utilized to input the amino acid sequence of the single-chain antibody, and the online analysis tool is used for calculating and predicting the physicochemical properties of the single-chain antibody, such as the amino acid quantity, the molecular formula, the molecular weight, the theoretical isoelectric point, the stability and the like. An online tool (http:// www.imgt.org/IMGT _ vquest/vquest) of an IMGT database of an international immune genetic information system is set as a mouse species, Immunoglobulin (IG) and single-chain antibody options, an anti-single-chain antibody nucleic acid sequence is submitted, and information such as allele, framework region and CDR region mark, and the like of a single-chain antibody heavy-light chain variable region encoding gene fragment is analyzed.

The test uses an online analysis tool of ExPASy to submit a single-chain antibody nucleic acid sequence, and the single-chain antibody obtained by calculation consists of 280 amino acids, has a molecular formula of C1327H2040N366O404S10, has a molecular weight of 29905.61, and has a theoretical isoelectric point of 8.95. The total number of negatively charged amino acid (Asp + Glu) residues was 17 and the total number of positively charged amino acid (Arg + Lys) residues was 22. The antibody is theoretically 30h in reticulocytes of mammals, more than 20h in yeast cells and more than 10h in escherichia coli. Instability index 55.58, classified as unstable protein. The aliphatic amino acid coefficient is 69.39, the average hydrophilic index is-0.239, and the protein is hydrophilic protein.

Analysis after submitting single chain antibody nucleic acid sequences to IMGT databases indicated. The VH 369bp at the 5 'end and the V kappa at the 3' end are 321bp in length. VH and V κ were linked by a 45bp linker sequence. The terminal also has an 18bp His tag. The VH consists of a V, D, J gene fragment, where the most consistent allele with the V gene fragment is Musmus IGHV1-18 x 01F or Musmus IGHV1-22 x 01F, with an identity of 89.93%. The allele with the highest identity to the J gene fragment was Musmus IGHJ1 × 01F or Musmus IGHJ1 × 03F (identity 90.57%), and the allele with the highest identity to the D gene fragment was Musmus IGHD1-1 × 01F. IMGT labels VH with 4 framework regions of amino acids 25-17-38-11 in length and VH with 3 CDR regions of amino acids 8-8-16 in length.

Vk is encoded by the V, J gene fragment, and the most consistent alleles were Musmus IGKV4-72 × 01F (94.57%) and Musmus IGKJ1 × 01F (100%). The amino acid length of V kappa 4 framework regions marked by IMGT is 26-17-36-10, and the amino acid length of three CDR regions of V kappa is 5-3-9.

Example 3 soluble expression experiment of pyrethroid pesticide metabolite broad-spectrum recognition Single-chain antibody

The specific steps of the embodiment are as follows:

mu.L of the glycerol stock solution of the single-chain antibody strain prepared in example 1 and having the amino acid sequence shown in SEQ ID NO.1, which had been frozen at 50 ℃ to 80 ℃, was added with 5mL of 2 XTY (containing 1% glucose and 50. mu.g/mL of kanamycin), and cultured overnight at 37 ℃ with shaking at 250 rpm. The next day, 2.5mL of the overnight culture was added to a 250mL 1L Erlenmeyer flask containing 2 XTY (final concentration 0.1% glucose and 50. mu.g/mL kanamycin), and the mixture was shake-cultured at 37 ℃ and 250rpm until OD600 became 0.9 (about 3 h). Adding IPTG of 0.25mM final concentration, culturing at 16 deg.C and 250rpm with shaking, and culturing for 8 h. The culture broth was centrifuged at 1800g for 10min at 4 ℃ and the supernatant was discarded. The remaining bacteria were resuspended in 125mL PBS, vortexed, and sonicated in an ice water bath. The ultrasonic conditions are as follows: the output power is 30%, the temperature is 25 ℃, the mode is 06, the working time is 30min, the ultrasound is 3s, and the interval is 4 s. Centrifuging at 4 deg.C for 30min at 10000g, and collecting supernatant to obtain whole bacteria crushed supernatant (S2). The purified single-chain antibody was purified by using the single-chain antibody in HisTrap HP column S2, the purified single-chain antibody was obtained for SDS-PAGE analysis by the method described in GE, and the concentration of the purified protein was measured by the Bradford method (see the document: "Burgis RR, Doiher MP. guide for protein purification (second edition of original text) [ M ]. Beijing: scientific Press; 2013, 69-70).

The results are shown in FIG. 2, lane 1: pre column sample, lane 2: column top eluate, lane M: protein molecular weight Marker, lane 3:50mM imidazole elution fraction, lane 4:100mM imidazole elution fraction, lane 5:200mM imidazole elution fraction. As can be seen, the test obtained electrophoretically pure single-chain antibody bands under the condition of 200mM imidazole elution, and the yield of the single-chain antibody was 2.68mg/L by calculating the protein concentration.

Example 4 detection application of pyrethroid pesticide metabolite broad-spectrum recognition single-chain antibody to 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol

In the experiment, competitive ELISA detection methods of 3-phenoxybenzoic acid (3-PBA), 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol are respectively established by using the purified single-chain antibody obtained in the embodiment 3, and the specific steps are as follows:

the PBA-OVA coating is diluted to 2 mu g/mL by CBS buffer solution, 100 mu L/well is added to an enzyme label plate, the temperature is 4 ℃ overnight, and the plate is washed by PBST for3 times. Add 2% MPBS to 200. mu.L/well, incubate at 37 ℃ for 1h, wash plate 3 times with PBST. mu.L of purified single-chain antibody (PBS diluted) diluted 40-fold and 50. mu.L of 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde or 3-phenoxybenzyl alcohol standards (10% methanol-PBS solubilized) were added to each well. The plate was washed 3 times with PBST after 1h incubation at 37 ℃. HRP-labeled anti-His monoclonal antibody diluted by PBS at the ratio of 1:5000 times is added into an ELISA plate at the concentration of 100 mu L/hole, the plate is bathed at 37 ℃ for 1h, and the PBST is washed for3 times. The substrate solution (10mL of CPBS buffer added with 100. mu.L of 10mg/mL TMB dissolved in dimethyl sulfoxide and 25. mu.L of 0.65% H) was prepared ₂ O ₂ ) Adding 100 mu L/hole enzyme label plate, standing at 37 ℃ for color development for 15 min. 2M H ₂ SO ₄ Add 50. mu.L/well quickly to the microplate and read the absorbance at 450 nm.

The logarithm of the concentration of 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde or 3-phenoxybenzyl alcohol is respectively used for corresponding B/B ₀ Plotting the value (ratio of absorbance value corresponding to standard substance to absorbance value corresponding to zero concentration standard substance) to draw standard inhibition curve, performing regression analysis with ELISA module of Graphpad Prism software, and calculating concentration I in inhibition ₅₀ Minimum detection Limit I ₁₀ And linear detection range (I) ₂₀ -I ₈₀ )。

The standard curve establishment method is a conventional technique in the art, and is reported in the documents "Liu Yuan, Zhang Cun-Zhen, Yu Xian-yang, Zhang Zhi-yong, Zhang Xiao, Liu Rong-Rong, Liu Xian-jin, Gong Zhen-min.Establment and evaluation of immunology for Zeranol in bovine root.journal of Zhejiang University Science B.2007, 8.: 900-.

The detection results are shown in FIG. 3, B/B ₀ Represents the ratio of the corresponding light absorption value of the 3-PBA standard product to the corresponding light absorption value of the zero-concentration standard product. Calculating the final inhibitory concentration (I) of 3-phenoxybenzoic acid to the single-chain antibody by regression analysis of a standard curve ₅₀ ) 0.55 mu g/mL and the lowest detection limit I ₁₀ 0.05. mu.g/mL, linear detection range (I) ₂₀ -I ₈₀ ) Between 0.15 and 2.64. mu.g/mL. Additional 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol on Single chain antibody I ₅₀ Respectively 0.59 and 0.63 mug/mL, and the single-chain antibody has similar recognition capability to 3-phenoxyformic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol, and has broad-spectrum specificity.

Sequence listing

<110> agricultural science and academy of Jiangsu province

Broad-spectrum recognition single-chain antibody of <120> pyrethroid pesticide metabolite and application thereof

<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

1. The broad-spectrum recognition single-chain antibody of the pyrethroid pesticide metabolite is characterized in that the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 1;

the pyrethroid pesticide is one or more of 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol.

2. The single chain antibody of claim 1, wherein said single chain antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.2 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 3.

3. The single chain antibody of claim 1, wherein the nucleotide sequence of said single chain antibody is set forth in SEQ ID No. 4.

4. Use of a single-chain antibody according to any one of claims 1 to 3 in the preparation of a test reagent for the detection of pyrethroid pesticide metabolites.