CN114644712A

CN114644712A - Method and kit for detecting procymidone

Info

Publication number: CN114644712A
Application number: CN202111592864.3A
Authority: CN
Inventors: 徐振林; 何晓婷; 王弘; 沈玉栋; 罗林; 陈子键; 雷红涛; 杨金易
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-06-21
Anticipated expiration: 2041-12-23
Also published as: CN114644712B

Abstract

The invention discloses a method and a kit for detecting procymidone, and establishes a method for detecting procymidone by using an anti-procymidone nano antibody with an amino acid sequence shown as SEQ ID NO.1 and develops a kit for detecting procymidone based on the method. The nano antibody prepared by the invention can be applied to the detection of a practical sample of the procymidone residue, has the advantages of high sensitivity, strong specificity, high temperature resistance, acid and alkali resistance, easy storage, low production cost, short production period and the like, and can be used as a novel material for detecting the procymidone. The method for preparing the nano antibody has universal applicability, can be used for screening and preparing other small molecular substance nano antibodies, and has higher application value.

Description

Method and kit for detecting procymidone

Technical Field

The invention relates to the technical field of detection of bactericides, in particular to a method and a kit for detecting procymidone.

Background

Procymidone (PRM) is a low-toxicity, cheap, high-efficiency and broad-spectrum systemic bisimide bactericide, has the action mechanism of inhibiting the synthesis of triglyceride in thalli, has special effect on botrytis and sclerotinia fungi, and is mainly used for preventing and treating gray mold and sclerotinia of fruit trees, vegetables and flowers and black spot of other crops or foods such as pseudo-ginseng, ginseng and the like. Although the toxicity of the procymidone is not high, the residue of the procymidone in fruits and vegetables can enter animals or human bodies through food chains, and the dichloroaniline metabolite of the procymidone can generate certain toxicity to the organisms, and can cause deformity, atrophy, lesion and the like of sexual organs of mammals when being serious. In recent years, due to the wide use of the procymidone on fruits and vegetables, the procymidone becomes one of pesticide varieties with higher detection rate and superstandard rate of pesticide residues, and particularly, the event that the procymidone exceeds the standard in the Chinese chives is subjected to medium-frequency exposure in supervision and spot inspection in various places, so that the enhancement of the detection of the procymidone pesticide residues is very necessary.

At present, most of methods for detecting procymidone residues are gas chromatography, gas chromatography-mass spectrometry and liquid chromatography. Although these instrumental methods have the advantages of accurate and reliable experiment, high sensitivity, good repeatability and the like, the defects of complex sample pretreatment process, long detection time, expensive instruments and the like exist, and the method can only be used for sampling detection of small-batch samples and cannot meet the requirements of on-site and rapid detection of large-batch samples.

The enzyme-linked immunoassay method based on the specific reaction of the antigen and the antibody has the advantages of high flux, high sensitivity, rapidness, easy operation, low cost and the like, can be prepared into a commercialized kit for on-site detection, and is widely applied to detection of pesticide and veterinary drug residues and trace toxins. The key to the success of the immunoassay method is to prepare antibodies with high sensitivity and specificity, monoclonal antibodies and polyclonal antibodies are most widely applied at present, but the traditional antibodies have the defects of long development period, low stability, harsh storage conditions and the like, and the prepared stable antibody with strong specificity and high sensitivity to procymidone has important significance for establishing a rapid, sensitive and accurate procymidone detection method.

The nano antibody (VHH) is a novel genetically engineered antibody, is derived from the Variable region of heavy chain antibodies in camels and sharks, and has the characteristics of low immunogenicity, high stability, high affinity, high specificity and the like. Compared with the conventional antibody, the nano antibody is easier to store and transport, and can be effectively refolded and restore the antigen affinity even after being denatured under extreme conditions such as high temperature, chemistry and pressure. In addition, the nano antibody can be expressed in a large amount by prokaryotes, the preparation period is short, the problem of batch difference of the traditional antibody can be effectively avoided, and the method has higher economic benefit and application advantage.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing method for detecting procymidone and provides a procymidone nano antibody and a preparation method and application thereof.

The first purpose of the invention is to provide a nano antibody against procymidone.

The second purpose of the invention is to provide a gene for coding the nano antibody.

The third object of the present invention is to provide a recombinant vector containing the nucleotide sequence of the gene according to the second object of the present invention.

The fourth object of the present invention is to provide a recombinant cell transfected with the expression vector of the third object of the present invention.

The fifth purpose of the invention is to provide the application of one or more of the nano antibody, the gene, the recombinant vector or the recombinant cell in the immunological detection analysis for detecting procymidone.

The sixth purpose of the invention is to provide a method for detecting procymidone.

The seventh purpose of the invention is to provide a kit for detecting procymidone.

In order to achieve the purpose, the invention is realized by the following scheme:

the method comprises the steps of constructing a bactrian camel immune antibody library, coating a detection antigen solid phase on an enzyme standard plate by using a phage display technology, and putting the bactrian camel immune antibody library for affinity panning to obtain a nano antibody specifically combined with the anti-putrefaction, wherein the amino acid sequence of the nano antibody is shown in SEQ ID No. 1. The method is applied to the immunological detection and analysis of the procymidone, and a rapid, sensitive and stable method for detecting the procymidone is established through the immunological detection and analysis.

The invention therefore claims the following:

the amino acid sequence of the nano antibody for specifically recognizing the procymidone is shown as SEQ ID No. 1.

Wherein, the framework regions (FR1-FR4) of the amino acid sequence of the nano antibody are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in sequence; the complementarity determining regions (CDR1-CDR3) are shown in sequence as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.

The nucleotide sequence of the gene for coding the specific recognition procymidone nano antibody is shown as SEQ ID NO. 9.

Specifically, the method comprises the following steps:

the amino acid sequence of VHH of the nano antibody is shown in SEQ ID NO. 1:

EVQLLDSGGGSVQAGGSLRLSCEVFTSKIATGYMGWFRQAPGKEREAVAITHTGSG NPWYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTCAAREAGSWELRRLSPEWY TYWGQGTLVTVSS；

the amino acid of the framework region FR1 of the nano antibody is shown in SEQ ID NO. 2:

EVQLLDSGGGSVQAGGSLRLSCEVF；

the amino acid of the framework region FR2 of the nano antibody is shown in SEQ ID NO. 3:

MGWFRQAPGKEREAVAI；

the amino acid of the framework region FR3 of the nano antibody is shown in SEQ ID NO. 4:

WYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTC；

the amino acid of the framework region FR4 of the nano antibody is shown in SEQ ID NO. 5:

WGQGTLVTVSS；

the amino acid of the complementarity determining region CDR1 of the nano antibody is shown in SEQ ID NO. 6:

TSKIATGY；

the amino acid of the complementarity determining region CDR2 of the nanobody is shown in SEQ ID NO. 7:

THTGSGNP；

the amino acid of the complementarity determining region CDR3 of the nanobody is shown in SEQ ID NO. 8:

AAREAGSWELRRLSPEWYTY；

the nucleotide sequence of the gene for coding the specific nano antibody is shown as SEQ ID NO. 9:

GAGGTGCAGCTGCTGGATTCTGGGGGAGGCTCGGTACAGGCTGGAGGGTCTCT GAGACTCTCGTGTGAAGTTTTTACAAGCAAGATAGCAACCGGCTACATGGGCTGGTT CCGGCAGGCTCCGGGTAAGGAGCGCGAGGCCGTCGCAATTACTCATACCGGTAGTGG AAACCCATGGTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCAAGACAACGC CAAGCTCACGGTGTATCTGCAAATGAACAACCTGAGACCTGAGGACACTGCCACGT ACACCTGTGCGGCCCGGGAGGCAGGGTCTTGGGAGCTCAGGCGGTTATCTCCAGAA TGGTATACCTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA。

a recombinant vector connected with the coding gene.

A recombinant cell comprising the expression vector, or capable of expressing the nanobody.

The application of one or more of the nano antibody, the gene, the recombinant vector and/or the recombinant cell in the detection of procymidone and/or the preparation of a procymidone immunological detection kit also belongs to the protection scope of the invention.

The invention claims a non-diagnostic purpose detection method of procymidone, which utilizes the nano antibody.

Preferably, the detection is carried out based on an indirect competitive ELISA method, a procymidone complete antigen obtained by coupling the procymidone hapten and carrier protein shown in the formula (I) is used as a detection antigen, the nano antibody is used as a detection antibody for detection,

preferably, the carrier protein is Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), or chicken egg albumin (OVA).

More preferably, the carrier protein is Bovine Serum Albumin (BSA), the detection antigen has a structural formula shown in (III),

more preferably, the solid phase carrier coated with the detection antigen is added with a sample to be detected and the nano antibody with the amino acid sequence shown as SEQ ID NO.1 in sequence, and after full reaction, the liquid is discarded and washed; adding enzyme labeled anti-diabody, fully reacting, discarding liquid, washing, fully reacting, discarding liquid, and washing; carrying out color development reaction and stopping the reaction; read 450nm OD values.

In a specific embodiment, the TMB developing solution in which developing solution A and developing solution B are mixed undergoes a developing reaction with 10% H₂SO₄(v/v) the reaction was terminated.

The invention also claims a kit for detecting procymidone, which contains the nano antibody.

Preferably, the method also comprises the step of taking a procymidone complete antibody obtained by coupling the procymidone hapten and a carrier protein as a detection antigen, taking the nano antibody as a detection antibody,

more preferably, the carrier protein is Bovine Serum Albumin (BSA), the structural formula of the detection antigen is shown as (III),

preferably, the detection antigen is coated on a solid support.

Preferably, the kit further comprises an enzyme-labeled secondary antibody, a color developing agent and a terminator.

More preferably, the color-developing agents are color-developing liquid a and color-developing liquid B.

More preferably, the terminating agent is 10% H₂SO₄(v/v)。

More preferably, the enzyme-labeled secondary antibody is an Anti-VHH-HRP secondary antibody.

Compared with the prior art, the invention has the following beneficial effects:

the nano antibody prepared by the invention can be prepared in a large scale by a gene engineering recombinant expression mode. The gene engineering recombination expression mode is to clone the gene coding the nano antibody to an expression vector and carry out mass preparation of the nano antibody in a protein expression mode. After the nano antibody is expressed by prokaryotes, immunological detection and analysis are carried out in the form of protein.

The nano antibody prepared by the invention can be applied to the detection of practical samples of procymidone residues. The nano antibody has the advantages of high sensitivity, strong specificity, high temperature resistance, acid and alkali resistance, easy preservation, low production cost, short production period and the like, and can be used as a novel material for detecting procymidone. The method for preparing the nano antibody has universal applicability, can be used for screening and preparing other small molecular substance nano antibodies, and has higher application value.

Drawings

FIG. 1 is a UV scanning spectrum of Pythium hapten, carrier protein and complete antigen.

FIG. 2 is a schematic representation of ELISA identification of Pythium positive clones.

FIG. 3 is an SDS-PAGE picture of a procymidone nanobody NbFM 5.

Fig. 4 is an optimization of ELISA antigen-antibody working concentration based on nanobody NbFM 5.

Fig. 5 is an indirect competition ELISA standard curve established based on nanobody NbFM 5.

Fig. 6 is a schematic diagram of the activity of nanobody NbFM5 at different concentrations of methanol/acetonitrile/acetone.

Fig. 7 is a schematic diagram of the activity of the nanobody NbFM5 after incubation at 70 ℃ for various times.

Fig. 8 is a schematic diagram of the activity of nanobody NbFM5 in PBS of different pH.

Fig. 9 is a schematic diagram of the activity of nanobody NbFM5 stored at different temperatures for different periods of time.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 preparation of Immunity antigen PRM-LF and detection antigen PRM-BSA

The design and synthesis of the procymidone hapten are completed by early work of a laboratory, and the structural formula of the procymidone hapten is shown as (I) (the compound is disclosed in CN 201910194037-a procymidone hapten and a synthesis method and application thereof):

first, experiment method

1. Preparation of complete antigen PRM-LF

The pythium hapten with the structural formula shown in (I) and Lactoferrin (Lactoferrin, LF) are coupled to form an immune antigen through an active ester method, the structural formula is shown in (II),

the specific operation method comprises the following steps: 2.876mg EDC and 1.725mg NHS were weighed out and 0.1mL DMF was added to dissolve the solids. And adding 3.518mg of the procymidone hapten into the mixture solution, and stirring at room temperature in the dark or shaking for 4 hours to activate the hapten. 20mg of LF was weighed, and 2mL of carbonate buffer (pH 9.4) was added to prepare a 10mg/mL protein solution, which was cooled in an ice bath. Dropwise adding the activated procymidone hapten into a stirring ice bath, and stirring the solution at room temperature overnight at the alkaline range by using a pH test paper. Dialyzing with PBS 3 times, subpackaging, and freezing at-20 deg.C. And scanning coupling raw materials (hapten and carrier protein LF) and coupling products by using an ultraviolet spectrophotometry, and judging whether coupling is successful or not by using an ultraviolet absorption spectrum obtained by scanning.

2. Preparation of complete antigen PRM-BSA

Coupling the semi-antigen of procymidone with the structural formula shown as (I) and Bovine serum albumin (Bovine serum mum alb mum, BSA) by an active ester method to form an artificial detection antigen, wherein the structural formula is shown as (III),

the specific operation method comprises the following steps: 6.535mg EDC and 3.92mg NHS were weighed out and 0.15mL DMF was added to dissolve the solids. 7.995mg of the procymidone hapten is added into the mixture solution, and the mixture solution is stirred at room temperature in the dark or is shaken for reaction for 4 hours to activate the hapten. 50mg of BSA was weighed and added to 5mL of carbonate buffer (pH 9.4) to prepare a 10mg/mL protein solution, which was cooled in an ice bath. Activated hapten is dropwise added into a stirring ice bath, a pH test paper is used for determining that the solution is in an alkaline range, and the solution is stirred at room temperature overnight. Dialyzing with PBS for 3 times, subpackaging, and freezing at-20 deg.C. Dialyzing with PBS for 3 times, subpackaging, and freezing at-20 deg.C. Scanning coupling raw materials (hapten and carrier protein BSA) and coupling products by using an ultraviolet spectrophotometry, and judging whether coupling is successful or not by using an ultraviolet absorption spectrum obtained by scanning.

Second, experimental results

The UV scanning spectrum of the Pythium hapten, carrier protein (LF, BSA) and coupling product is shown in FIG. 1. The characteristic peaks of the procymidone hapten are positioned at 210nm and 280nm, the LF ultraviolet absorption peaks are positioned at 210nm and 280nm, and the BSA ultraviolet absorption peaks are positioned at 228nm and 280 nm. Ultraviolet absorption peaks of complete antigen PRM-LF II after coupling of carrier protein LF and procymidone hapten are 229nm and 280nm, and ultraviolet absorption peaks of complete antigen PRM-BSA III after coupling of carrier protein BSA and procymidone hapten are 242nm and 278 nm. Therefore, ultraviolet absorption peaks of the two artificial antigens are obviously shifted relative to hapten and carrier protein, and the absorbance is changed, which shows that coupling of the procymidone hapten and the carrier protein LF and BSA is successfully realized.

Example 2 construction of Bactrian camel immune antibody library

1. Bactrian camel immunization protocol

Animal immunization is carried out on healthy bactrian camel, the complete antigen PRM-LF with the structural formula shown in (II) prepared in example 1 is used as an immunizing antigen, subcutaneous injection is carried out on the neck of the bactrian camel, and the immunizing dose is 0.5mg for each time. The first immunization is mixed and emulsified with 0.5mL of complete Freund's adjuvant and immune antigen and then used for immunization, and the subsequent boosting immunization is emulsified with 0.5mL of incomplete Freund's adjuvant and antigen and then immunized, wherein 4 boosting immunizations are carried out at an interval of 2 weeks. And after the fourth immunization and the fifth immunization, collecting 50-100 mL of peripheral blood for constructing a nano antibody library.

2. Isolation of Bactrian camel lymphocytes

The method for collecting bactrian camel peripheral blood needs to separate lymphocytes as soon as possible, and comprises the following specific operation methods: mixing and diluting bactrian camel peripheral blood and sterile normal saline in a clean container without RNase in equal volume. The diluted peripheral blood was centrifuged with a commercial lymphocyte separation medium, and blood cells of different densities were centrifuged to distribute the lymphocyte separation medium at different depths, wherein the lymphocytes formed a white cell layer at a depth of about 1/3 below the surface of the liquid. At room temperature, 850g, centrifuge for 25 min. Carefully collecting the cell pellet in an enzyme-inactivated clean container by using a rubber-tip dropper or a pipette without RNase, washing the redundant lymphocyte separation solution on the surface of the cell by using sterile physiological saline, and centrifugally collecting the cell pellet. Adding lysis solution TRNsol into the collected lymphocytes, repeatedly blowing and beating with a pipette or a rubber head dropper without RNase until the lymphocytes are completely lysed in the TRNsol, and storing at-80 deg.C for later use.

3. Extraction of Total RNA

Extracting total RNA from the lymphocytes preserved in the TRNsol lysate by using a commercial RNA extraction kit or other effective RNA extraction methods, and performing operation according to the instruction of the RNA extraction kit. After extraction of total RNA, a small sample was subjected to nucleic acid electrophoresis and the RNA concentration was measured in a ultramicrospectrophotometer (nanodrop).

4. Synthesis of cDNA

And (3) carrying out reverse transcription on mRNA in the RNA sample obtained in the last step into cDNA by adopting a commercial reverse transcription kit, and carrying out operation according to the instruction. After the reverse transcription is finished, the products are firstly mixed uniformly, then are packaged in different sterile centrifuge tubes, and are stored in an environment of minus 80 ℃.

5. Amplification of nano antibody target gene VHH

Performing two-step amplification by adopting nested PCR to obtain a target gene VHH of the nano antibody, wherein the sequences of the used primers are shown in a table 1:

TABLE 1 Bactrian camel heavy chain antibody Gene primer sequences

The first round of PCR uses cDNA as its PCR template, and the specific reaction parameters are shown in Table 2:

TABLE 2 nested PCR first step reaction System and reaction conditions

The PCR product of the first step can generate two product bands of 1000bp and 750bp after nucleic acid electrophoresis, the 750bp band is cut and recovered, and the concentration is measured.

Second round PCR the second round PCR amplification was performed using the recovered product of the first round PCR as a template, and the specific reaction parameters are shown in Table 3:

TABLE 3 nested PCR second step reaction System and reaction conditions

6. Gene library construction

(1) Enzyme digestion of target gene VHH and vector pComb3xss

Carrying out enzyme digestion reaction on the VHH target gene and the pComb3xss vector by adopting Sfi I enzyme. Enzyme cutting conditions are as follows: reacting for 16h at constant temperature of 50 ℃.

Recovering a band with the molecular weight of 3500bp from the enzyme digestion product of the pComb3xss vector through agarose gel; the VHH gene enzyme cutting product is directly cleaned and recovered through a DNA recovery kit.

(2) Ligation of the cleavage products

The vector pComb3xss and VHH fragments are mixed evenly (molar ratio is 1: 3), reacted for 16h at 16 ℃, and then cleaned and recovered by a DNA recovery kit.

(3) Electric shock conversion

Adding 5 mu L of the ligation product into 50 mu L of electrotransformation competent E.coil TG1, gently mixing uniformly, transferring into an electric rotating cup of 0.1cm for electric shock transformation (voltage is 1.8kv), immediately adding 950 mu L of SOC culture medium preheated to 37 ℃ into the electric rotating cup after electric shock, and shaking bacteria at 250rpm at 37 ℃ for 1h to recover cells.

100 mu L of resuscitating bacteria liquid is taken for gradient dilution, 100 mu L of each concentration gradient diluted bacteria liquid is taken and coated on an LB-Amp culture dish with the diameter of 90 mm as a counting plate, and the bacteria liquid is cultured overnight at 37 ℃. And (3) coating all the rest undiluted resuscitation bacteria liquid on LB-Amp culture dishes with the diameter of 120mm, coating 2-3 culture dishes per 1mL bacteria liquid as an amplification plate, and performing amplification culture at 37 ℃ overnight.

Counting the number of bacterial colonies on the counting culture dish, calculating the total number of bacteria in the resuscitation bacteria liquid, and carrying out multiple electric shock transformation to accumulate the total number of transformed bacterial coloniesThe meter reaches 10⁷More than cfu, the number is the library capacity of the nano antibody gene library.

Scraping the transgenic escherichia coli colonies in the amplification plate by using a cell scraper, uniformly mixing, adding glycerol with the final concentration of 25%, taking 50 mu L of bacterial liquid, performing gradient dilution to determine the cell number, subpackaging the rest bacterial liquid, and freezing and storing at-80 ℃ to obtain the procymidone nano antibody gene library.

7. Phage rescue

Inoculating cells with more than 10 times of library capacity into 200mL LB-Amp according to the cell number measuring result of the transgenic escherichia coli, and culturing at 37 ℃ and 250rpm until logarithmic phase; 1mL of the mixture was added to the flask with a titer of 10¹²The helper phage M13K07 was left to stand at 37 ℃ for 30min at cfu/mL or more, and then cultured at 250rpm for 1h, and kanamycin (70. mu.g/mL) was added thereto and cultured at 37 ℃ overnight at 250 rpm. Centrifuging at 12000rpm at 4 ℃ for 15min, taking the supernatant, adding 1/4 volumes of PEG/NaCl solution (100g of PEG 8000 and 73.05g of sodium chloride are added with water to be constant volume to 500mL), and carrying out ice bath for 2-3 h. Centrifugation was carried out at 12000rpm for 15min at 4 ℃ and the supernatant was discarded, the pellet was resuspended in 1mL TBS, transferred to a 2mL centrifuge tube, centrifuged at 12000rpm for 5min at 4 ℃ and filtered through a 0.22 μm polyethersulfone filter. And (3) taking 10 mu L of phage to determine titer, adding glycerol with the final concentration of 50 percent into the rest, and preserving at minus 80 ℃ to obtain the procymidone nano antibody phage library.

Example 3 affinity panning and identification of Nanobodies

First, experiment method

1. Affinity panning of Nanobodies

(1) Detection of antigen immobilization

Coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the complete antigen PRM-BSA with the structural formula shown in (III) prepared in example 1 to 1 mu g/mL, adding the diluted complete antigen PRM-BSA into micropores of an ELISA plate, keeping the diluted complete antigen PRM-BSA in each hole at 100 mu L, and standing the diluted complete antigen BSA at 4 ℃ overnight. The following day, after washing the plate twice with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20), 150. mu.L of 1% BSA-PBS (w/v) solution was added to each well and allowed to stand at 37 ℃ for 2 hours. The well was decanted, patted dry on absorbent paper and stored at 4 ℃ after 1h at 37 ℃ until use.

(2) Positive phage selection

BSA was added to the above Pythium nanobody phage library to make the final concentration of BSA 1% (w/v), phage cells were added to 3 microwells with immobilized antigen, 100. mu.L of each well, and incubated at 37 ℃ for 1 h. Free phage in wells were discarded, wells were washed 10 times with PBST and 5 times with PBS.

And adding a procymidone standard substance with the concentration of 5 mu g/mL into each hole, performing shaking incubation for 4 hours at 37 ℃ for performing competitive reaction by using 100 mu L of each hole, and collecting liquid in the micropores into a sterile centrifuge tube. The phage at this time is called "competitive output". To prevent loss of diversity, Trypsin (10mg/mL) was added to the wells where the competitive elution was completed and eluted for 30min, and the liquid in the microwells was collected in sterile centrifuge tubes, called "enzyme wash output", and the first round of screening was completed.

The phage titer is determined before and after screening, and the specific operation method is as follows:

10 mu.L of two output phages are respectively diluted by 10-fold gradient, and the expected titer and two adjacent dilutions in front and back are selected (the input dilution is 10 times in general)⁷、10⁸、10⁹The output dilution factor is 10²、10³、10⁴) 10 mul of diluent is taken to infect E.coil TG1 in log phase of 100 mul, and is kept still for 30min at 37 ℃, an LB-Agar-Amp plate is coated, and colony counting and phage titer calculation are carried out after overnight culture, and the calculation formula is as follows:

(3) phage amplification

After the screened phage is amplified, the phage is used for the next screening, and the method for amplifying the phage is as follows:

half of the competing eluted output phages were added to 4mL log phase e.coil TG1 and left for 30 min. LB medium was added to a total volume of 10mL and Amp was added to working concentration (10. mu.g/mL), shaken at 37 ℃ 250rpm for 30min, 50. mu.L was added to a titer of 10¹²cfu/mL M13KO7 helper phage, standing for 30min, shaking at 37 deg.C and 250rpmFor 1h, LB was added to a total volume of 100mL, Amp and Kana were added, and the mixture was shaken at 37 ℃ and 250rpm overnight.

Centrifuging the bacterial liquid, transferring the supernatant into a new centrifugal bottle, adding 1/4 volumes of mixed solution of PEG and NaCl (100g of PEG 8000 and 73.05g of sodium chloride are added with water to a constant volume of 500mL), mixing uniformly, carrying out ice bath for 2h, centrifuging at 12000rpm for 20min, discarding the supernatant, reversing the supernatant on absorbent paper to remove residual moisture as much as possible, resuspending the white precipitate with 1mL of TBS containing 1% BSA (w/v), passing through a 0.22 mu m polyether sulfone filter membrane, storing at-20 ℃, serving as next round of screening input, and repeating the screening scheme of the step (2).

The screening steps are carried out for 4 rounds in total, the concentration of the antigen detected in each round is fixed to be 1ug/mL and is unchanged, and the concentrations of the drugs used in the competitive reactions of the 2 nd, 3 rd and 4 th rounds are respectively reduced to 2000ng/mL, 400ng/mL and 40 ng/mL.

2. Identification of Positive clones

And (3) adopting an indirect enzyme-linked immunosorbent assay to carry out the identification of the positive phage clone. The specific method comprises the following steps:

(1) immobilization of antigens

Coating solution for detecting antigen (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Water was added to a constant volume of 250mL) to 1. mu.g/mL, and the mixture was allowed to stand at 4 ℃ overnight. Washing twice with 20 times diluted washing solution PBST (0.01M PBS, 0.05% Tween-20) the next day, adding 2% skimmed milk powder (w/v) with PBS, adding 150 μ L into each well, sealing at 37 deg.C for 2 hr, discarding the sealing solution, patting, oven drying at 37 deg.C for 60min, and storing at 4 deg.C for use.

(2) Nanobody miniexpression

Third, enzyme washing of four rounds of panning and competitive elution output titer determination plate randomly selected 96 single colonies, inoculated into each well of 0.5mL LB-Amp 96 well plate, simultaneously inoculated with a phage infection of E.coil TG1 single colony as negative control, 37 degrees C culture overnight, as the bacterial liquid "mother board".

Taking 10 mu L of bacterial liquid from each hole of the mother plate, inoculating the bacterial liquid into another 96-hole deep-hole plate with 1mL of LB-Amp in each hole, wherein the number of the inoculated hole corresponds to the mother plate, culturing for 3h at 37 ℃ and 180rpm, adding IPTG into each hole to ensure that the final working concentration is 1mM, culturing at 37 ℃ and 180rpm overnight, and storing the mother plate at 4 ℃ for later use.

(3) Enzyme-linked immunoassay positive clone

Centrifuging a deep-hole plate at 4000rpm for 20min, taking two enzyme-labeled plates with immobilized antigens, taking the plate 1 as a titer plate for detecting antibodies, and adding 50 mu L of PBS into each hole; plate 2 was used as an inhibition plate, PBS containing 50ng/mL of a Pythium standard drug was added, and the supernatant was aspirated from the centrifuged 96-well plate, and 50. mu.L of each well was added to the corresponding numbered enzyme-labeled well. Incubation was carried out at 37 ℃ for 30min, and the wells were rinsed five times with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20) and the liquid in the wells was tapped off. Anti-VHH-HRP secondary antibody (GenScript, A01861-200) was diluted 5000-fold with PBST, 100. mu.L per well, and incubated at 37 ℃ for 30 min. Washing with 20 times diluted washing solution PBST (0.01M PBS, 0.05% Tween-20) for five times, drying the liquid in the well, adding 100 μ L of TMB developing solution (Solarbio, PR1210) mixed with developing solution A and B in advance at equal volume into each well, and developing at 37 deg.C for 10 min; adding 50 mu L of stop solution 10% H₂SO₄(v/v) termination reaction; the absorbance at 450nm was measured with a microplate reader.

The inhibition rate of each positive clone was calculated according to the following formula. Selecting clones which have OD values 3 times larger than those of the negative control holes and have obvious inhibition (the inhibition rate is more than 20%) in the plate 1, recording the numbers of the corresponding holes, transferring the bacterial liquid of the corresponding holes in the mother plate into a sterile centrifuge tube, and adding glycerol for freezing and storing for later use.

Second, experimental results

And (3) sending the strain of the nano antibody obtained by indirect competition ELISA identification to a sequencing company for gene sequencing, and obtaining the amino acid sequence of the nano antibody according to the DNA sequencing result and a codon table. The results showed that 7 strains of procymidone nanobodies with different sequences were obtained and named NbFM 1-NbFM 7 respectively. The results of identifying these seven positive clones are shown in fig. 2, and the inhibition ratios of NbFM 1-NbFM 7 were 78.91%, 74.68%, 88.61%, 44.01%, 93.57%, 38.02% and 36.19%, respectively, with NbFM5 being the most prominent in indirect competitive ELISA assay and the highest inhibition ratio.

NbFM5 has an amino acid sequence shown in SEQ ID NO.1, the nanobody comprises 4 FR framework regions and 3 CDR complementarity determining regions, and the arrangement sequence is FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, wherein the framework regions (FR1-FR4) are shown in sequence as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SE 1% Q ID NO. 5; the complementarity determining regions (CDR1-CDR3) are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, in that order.

Meanwhile, the nucleotide sequence of the gene for coding the NbFM5 is shown as SEQ ID NO. 9.

Specifically, the method comprises the following steps:

the amino acid sequence of VHH of the nano antibody is shown in SEQ ID NO. 1:

the amino acid of the framework region FR1 of the nano antibody is shown as SEQ ID NO. 2:

EVQLLDSGGGSVQAGGSLRLSCEVF；

MGWFRQAPGKEREAVAI；

WYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTC；

WGQGTLVTVSS；

the amino acid of the complementarity determining region CDR1 of the nanobody is shown in SEQ ID NO. 6:

TSKIATGY；

THTGSGNP；

the amino acid of the complementarity determining region CDR3 of the nano antibody is shown in SEQ ID NO. 8:

AAREAGSWELRRLSPEWYTY；

EXAMPLE 4 Mass production of Nanobody NbFM5

Bacterial strain plasmids of an expression vector carrying NbFM5 gene (the nucleotide sequence is shown as SEQ ID NO. 9) are extracted by a kit, transformed into competent E.coil BL21(DE3) and coated on an LB-Agar-Amp plate for culture to obtain single colonies. A single colony was inoculated into 10mL of LB-Amp, and cultured overnight at 37 ℃ and 250 rpm. The overnight culture was inoculated at 1% (v/v) into 100mL LB-Amp, incubated at 37 ℃ and 250rpm for log phase, and incubated overnight at 37 ℃ and 250rpm with IPTG added to a working concentration of 1 mM.

Centrifuging the bacterial liquid, removing the supernatant, and extracting antibody periplasmic cavity protein by using a sucrose osmotic pressure freeze-thaw method, wherein the specific method comprises the following steps: adding 3.75mL of TES solution for resuspension and precipitation, freezing at-80 ℃ for more than 2h, melting in 37 ℃ water bath, adding 15mL of TES diluted by 5 times, shaking at 250rpm for 2h to fully extract protein, centrifuging to obtain supernatant, and purifying the supernatant by nickel-agarose gel affinity chromatography to obtain the nano antibody NbFM5 (the amino acid sequence is shown in SEQ ID NO. 1) shown in the figure 3.

Example 5 determination of working concentration and sensitivity of Nanobody NbFM5

First, experiment method

1. Immobilization of antigens

Coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL) to dilute the complete antigen PRM-BSA with the structural formula shown in (III) prepared in example 1 to a series of concentrations of 2000, 1000, 500, 250ng/mL and the like, and adding the complete antigen PRM-BSA into an ELISA plateIn wells, 100. mu.L of each well was allowed to stand overnight at 4 ℃. The following day, after washing the plate twice with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20), 150. mu.L of 1% BSA-PBS (w/v) solution was added to each well and allowed to stand at 37 ℃ for 2 hours. The liquid in the wells was poured out, patted dry on absorbent paper and stored at 4 ℃ after 1h at 37 ℃ for future use.

2. Determination of working concentration of antibody

The purified nano antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 is diluted by PBS to be respectively diluted into a series of working solutions with different concentrations. mu.L PBS per well was added to the microplate at different coating concentrations, and then 50. mu.L of the antibody after dilution was added in a gradient, and 3-well replicates were performed for each antibody concentration, while a blank control (100. mu.L PBS) was performed with 3 wells. Incubation was carried out at 37 ℃ for 30min, the wells were washed 5 times with 20-fold diluted wash PBST (0.01M PBS, 0.05% Tween-20), and 100. mu.L of Anti-VHH-HRP secondary antibody (GenScript, A01861-200) diluted 5000-fold with PBST was added to each well after blotting onto absorbent paper. Incubating at 37 deg.C for 30min, washing the wells with 20-fold diluted washing solution PBST (0.01M PBS, 0.05% Tween-20) for 5 times, drying on absorbent paper, adding 100 μ L of TMB developing solution (Solarbio, PR1210) mixed with equal volume of developing solution A and developing solution B into each well, incubating at 37 deg.C for 10min in the dark, adding 50 μ L of stop solution 10% H into each well₂SO₄(v/v), reading OD450nm on a microplate reader.

The antibody concentration of OD450nm between 1 and 1.5 is the working concentration of the nano antibody, and under the experimental conditions, the working concentrations of the nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 are 43.33, 43.33 and 54.17ng/mL respectively when the coating concentration is 2000, 1000, 500 and 250 ng/mL.

3. Indirect competitive ELISA standard curve drawing

The procymidone standards were diluted with PBS into a series of procymidone solutions of different concentrations, 50 μ L of procymidone of different concentrations was added to each well of the enzyme standard plate of different coating concentrations, 3-well replicates of each concentration were performed, and a 3-well drug blank (50 μ L PBS) was prepared. The nanometer antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 is respectively diluted to the working concentrations of 43.33, 43.33 and 54.17ng/mL by PBS,50 μ L of diluted antibody was added to wells of corresponding different coating concentrations. After incubation at 37 ℃ for 30min, the wells were washed 5 times with 20-fold diluted wash PBST (0.01M PBS, 0.05% Tween-20), and 100. mu.L of a 5000-fold Anti-VHH-HRP secondary antibody (GenScript, A01861-200) diluted with PBST was added to each well after blotting on absorbent paper. Incubating at 37 deg.C for 30min, washing the micropores with 20 times diluted washing solution PBST (0.01M PBS, 0.05% Tween-20) for 5 times, drying on absorbent paper, adding 100 μ L of TMB developing solution (Solambio, PR1210) mixed with equal volume of developing solution A and developing solution B, incubating at 37 deg.C for 10min in dark, adding 50 μ L of stop solution 10% H₂SO₄(v/v), reading OD450nm on a microplate reader.

The mean value of the OD450nm values of the drug blank was recorded as B₀The average OD450nm at different drug concentrations was designated B_xCalculating B by Excel under different drug concentrations_x/B₀Ratios and standard deviations for each set of parallel data. With the drug concentration as the abscissa, B_x/B₀And (4) drawing a scatter diagram in Origin software and fitting a logistic function to establish an indirect competition standard curve, wherein the ratio is a vertical coordinate. In addition, the obtained labeled parameters IC50, B of different coating concentrations₀、B₀The IC50 is compared to select the optimal working concentration of antigen and antibody.

Second, experimental results

The antigen-antibody working concentration of the indirect competitive ELISA method of the nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 is optimized by a chessboard titration method. As shown in FIG. 4, the IC50 value gradually decreased with the decrease in the concentration of the antigen to be detected, and B was found to be₀the/IC 50 value rose first and then fell, the IC50 value was lower compared to the B value at a coating concentration of 500ng/mL₀the/IC 50 was the greatest, so a coating concentration of 500ng/mL and an antibody concentration of 43.33ng/mL was chosen as the optimal antigen and antibody concentration for this ELISA method.

According to the selected antigen-antibody concentration, an indirect competition ELISA standard curve graph established based on a nano antibody NbFM5 with an amino acid sequence shown as SEQ ID NO.1 is shown in FIG. 5, and the standard curve is S-shaped, the linear correlation is good, the detection range is 0.19-2.38 ng/mL, the IC50 is 0.67ng/mL, the lowest limit of detection (LOD) is 0.13ng/mL, and the detection sensitivity is high.

Example 6A method for detecting procymidone

1. Pre-coating a plate: coating solution (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the complete antigen PRM-BSA with the structural formula shown in (III) prepared in example 1 to 500ng/mL, adding the diluted complete antigen PRM-BSA into micropores of an ELISA plate, keeping the diluted complete antigen PRM-BSA in each hole at 100 mu L, and standing the diluted complete antigen BSA at 4 ℃ overnight. The following day, after washing the plate twice with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20), 150. mu.L of 1% BSA-PBS (w/v) solution was added to each well and allowed to stand at 37 ℃ for 2 hours. The well was decanted, patted dry on absorbent paper and stored at 4 ℃ after 1h at 37 ℃ until use.

2. Marking standard blank well B₀Standard and sample wells, 3 well replicates were performed. In B ₀50 μ L of PBS was added, 50 μ L of standard solutions of different concentrations were added to each standard well, and 50 μ L of diluted sample solution was added to each sample well. The nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 was diluted to the working concentration of 43.33ng/mL with PBS, 50. mu.L of the diluted antibody solution was added to all the wells, and incubation was performed at 37 ℃ for 30 min.

3. The wells were washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20) and blotted dry on absorbent paper.

4. mu.L of Anti-VHH-HRP secondary antibody (GenScript, A01861-200) diluted 5000-fold with PBST was added to each well, incubated at 37 ℃ for 30min, and the wells were washed 5 times with 20-fold diluted wash PBST (0.01M PBS, 0.05% Tween-20) and blotted dry on absorbent paper.

5. mu.L of TMB developing solution (Solarbio, PR1210) mixed with equal volumes of developing solution A and developing solution B was added to each well, and incubated at 37 ℃ for 10min in the absence of light.

6. Add 50. mu.L of stop solution 10% H to each well₂SO₄(v/v), reading OD450nm on a microplate reader.

Thirdly, interpretation of results

The average value of blank OD450nm values of the standard is recorded as B₀OD450nm at different drug concentrations andthe average value of the holes to be measured of the sample is recorded as B_xCalculating B for different drug concentrations or sample wells_x/B₀Ratio and standard deviation of each set of parallel data. Taking the logarithm value of the concentration of the standard substance as an abscissa, B_x/B₀The ratio is the ordinate, and a standard curve graph is drawn. According to the average absorbance value of the sample hole, the abscissa of the corresponding point on the curve, namely the logarithm value of the concentration of the procymidone, can be obtained, and the obtained logarithm is the concentration of the procymidone in the measuring solution. Since the sample is pre-diluted, the concentration of the sample obtained from the standard curve is multiplied by the dilution factor.

Example 7 kit for detecting procymidone

Composition of kit

1. A detachable enzyme label plate pre-coated with BSA coupled procymidone antigen: coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the complete antigen PRM-BSA with the structural formula shown in (III) prepared in example 1 to 500ng/mL, adding the diluted complete antigen PRM-BSA into micropores of an ELISA plate, keeping the diluted complete antigen PRM-BSA in each hole at 100 mu L, and standing the diluted complete antigen BSA at 4 ℃ overnight. The following day, after washing the plate twice with a 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20), 150. mu.L of 1% BSA-PBS (w/v) solution was added to each well and allowed to stand at 37 ℃ for 1 hour. The well was decanted, patted dry on absorbent paper and baked at 37 ℃ for 1 h.

2. Enzyme labeling reagent: Anti-VHH-HRP secondary antibody, when used, needs to be diluted 5000 times.

3. And (3) standard substance: different concentrations of the procymidone standard solution.

4. Dilution of antibody with sample: PBS.

5. Dilution of labeled antibody: PBST.

6. A color developing liquid A and a color developing liquid B.

7. Stopping liquid: 10% (v/v) H₂SO₄。

8. Wash concentrated (20 ×): PBST (0.01M PBS, 0.05% Tween-20) was used for plate washing.

9. Detecting an antibody: the amino acid sequence is shown as NbFM5 of the nano antibody shown in SEQ ID NO. 1.

Secondly, use of the kit

Numbering in advance and marking blank holes B of standard products₀Standard and sample wells, 3 well replicates were performed. In B ₀50 μ L of PBS was added, 50 μ L of standard solutions of different concentrations were added to each standard well, and 50 μ L of diluted sample solution was added to each sample well. The nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 was diluted to the working concentration of 43.33ng/mL with PBS, 50. mu.L of the diluted antibody solution was added to all the wells, and incubation was performed at 37 ℃ for 30 min. The wells were washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20) and then 100. mu.L of enzyme-labeled secondary antibody was added to each well after blotting on absorbent paper. Incubating at 37 ℃ for 30min, washing the micropores 5 times by using washing liquor PBST (0.01M PBS, 0.05% Tween-20) diluted by 20 times, drying the micropores on absorbent paper, adding 100 mu L of TMB developing solution (Solambio, PR1210) mixed with equal volume of developing solution A and developing solution B into each hole, incubating for 10min in the dark at 37 ℃, adding 50 mu L of stop solution into each hole, and reading the OD value of 450nm on an enzyme-linked immunosorbent assay within 5 min.

Third, interpretation of results

The average value of blank OD450nm values of the standard is recorded as B₀The average value of the OD450nm and the sample to-be-measured holes at different drug concentrations is recorded as B_xCalculating B of different drug concentrations or sample wells by using Excel_x/B₀Ratios and standard deviations for each set of parallel data. Taking the logarithm value of the concentration of the standard substance as an abscissa, B_x/B₀The ratio is the ordinate, and a standard curve graph is drawn. According to the average absorbance value of the sample hole, the abscissa of the corresponding point on the curve can be obtained, namely the logarithm value of the concentration of the procymidone, and the antilog is obtained and is the concentration of the procymidone in the measuring liquid. Since the sample is pre-diluted, the concentration of the sample obtained from the standard curve is multiplied by the dilution factor.

Example 8 determination of the specificity of Nanobody NbFM5

First, experiment method

With the complete antigen PRM-BSA having the structural formula shown in (iii) prepared in example 1 as a detection antigen and the nanobody NbFM5 as a detection antibody, other 9 types of procymidone analogs (vinclozolin, dimethachlon, iprodione, folpet, dichlornasal, hexachlorophene) and procymidone metabolite (3, 5-dichloroaniline) standard solutions were prepared, and a standard curve of each procymidone analog and metabolite was drawn by the indirect competition ELISA method in example 5 using the kit of example 7, to obtain the respective IC50 values. The cross-reactivity of each drug with nanobody NbFM5 was calculated using the following formula:

second, experimental results

The results show that the cross reaction rates of the nano antibody NbFM5 and the Pythium metabolite 3, 5-dichloroaniline and analogues thereof, namely ethephon, dimethachlon, iprodione, folpet, dichlorodiphenylene and hexachlorocyclohexane are all lower than 0.1% (IC50 is more than 700 ng/mL); the nano antibody NbFM5 can specifically recognize the procymidone, and the detection method established by the invention has high specificity for procymidone detection.

Example 9 determination of the Activity of Nanobody NbFM5 in organic solvents of different concentrations

First, experiment method

Different volume ratios (0%, 20%, 40%, 60%, 80%, 100%) of methanol, acetonitrile, acetone solutions were prepared with PBS, respectively, and the procymidone standards were uniformly diluted to a drug concentration of 3ng/mL with an inhibition rate of about 80% on the standard curve established in example 5. The nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 was diluted to a working concentration of 43.33ng/mL with PBS. 50 mu L of standard product diluent prepared by organic solvents with different volume ratios is added into each well of the titer hole, and then 50 mu L of nano antibody solution with the amino acid sequence shown as SEQ ID NO.1 after dilution is added. 50 mu L of the procymidone standard solution diluted to 3ng/mL is added into each hole of the inhibition hole, and 50 mu L of the diluted nano antibody solution with the amino acid sequence shown as SEQ ID NO.1 is added.

The detection kit in example 7 was used to measure the binding capacity of the antibody to the antigen to be detected and the procymidone standard drug (3ng/mL) in different organic solvents, and the tolerance of the nano antibody NbFM5 with the amino acid sequence shown in SEQ ID No.1 to different organic solvents was evaluated.

Second, experimental results

Since the above antibody was diluted with PBS, the actual organic concentrations of the antibody were 0%, 10%, 20%, 30%, 40%, and 50%, respectively. The inhibition ratio can be calculated from the inhibition ratio formula of example 3. The determination result is shown in fig. 6, the binding activity of the nano antibody NbFM5 with the amino acid sequence shown in SEQ ID No.1 with the detection antigen and small molecules in low concentration methanol solution below 10% is less affected, and the binding activity of the antibody with the detection antigen can only be retained at 30% and 40% when the concentration of acetonitrile and acetone solution reaches 10%. In addition, the binding activity of NbFM5 to the test antigen and small molecule drug both decreased dramatically with increasing organic solvent concentration. When the proportion of methanol, acetonitrile and acetone in the organic solvent mixed solution reaches 40%, 20% and 20% respectively, the antibody is completely inactivated.

The nano antibody NbFM5 with the amino acid sequence shown in SEQ ID No.1 has poor tolerance to three organic solvents (methanol, acetonitrile and acetone) commonly used in the pretreatment of the standard method, and the organic solvent should be removed in the working solution of the antibody as much as possible to avoid the influence of high-concentration organic solvent on the sensitivity of the antibody.

EXAMPLE 10 determination of the Activity of Nanobody NbFM5 at different temperatures

First, experiment method

After the nano antibody NbFM5 with the PBS amino acid sequence shown in SEQ ID NO.1 is diluted to the working concentration (43.33 ng/mL), the nano antibody NbFM5 is incubated at high temperature of 70 ℃ for different periods of time (0, 10, 20, 30, 40, 50 and 60min), and then the Pythium standard is diluted by PBS to the drug concentration 3ng/mL with the inhibition rate of about 80% of the standard curve established in the example 5. Using the kit of example 7, the binding activity of the antibody to the test antigen and the Mucor standard drug (3ng/mL) after the high temperature treatment for various times was measured, respectively.

Second, experimental results

The inhibition rate can be calculated from the inhibition rate formula in the experimental method of example 3. The determination result is shown in fig. 7, the binding activity of the nanobody NbFM5 with the amino acid sequence shown in SEQ ID No.1 to the detection antigen and the binding activity of the small molecule analyte decrease with the increase of the incubation time, wherein the decrease of the binding activity of the nanobody to the small molecule analyte is relatively gentle. The nanobody NbFM5 having an amino acid sequence shown in SEQ ID No.1 still had 50% of antigen binding activity and 66% of inhibition rate even after heating at 70 ℃ for 1 hour. The nano antibody NbFM5 with the amino acid sequence shown in SEQ ID No.1 can be refolded to recover most of antigen binding capacity after high-temperature denaturation, and the characteristic can enable the nano antibody NbFM5 to have great application advantages in a high-temperature detection environment.

Example 11 determination of the Activity of Nanobody NbFM5 at different pH conditions

First, experiment method

The solvent of nanobody NbFM5, having the amino acid sequence shown in SEQ ID No.1, was diluted to a working concentration (43.33 ng/mL) and the procymidone standards were diluted to a drug concentration of 3ng/mL, with an inhibition rate of about 80% of the standard curve established in example 5, using 0.01M PBS at different pH values (2.4, 3.4, 5.4, 6.4, 7.4, 8.4, 9.4, 10.4) as diluents, respectively. The kit of example 7 was used to measure the binding capacity of the antibody to the test antigen and the procymidone standard drug (3ng/mL), and the binding activity of the nanobody NbFM5 with the amino acid sequence shown in SEQ ID No.1 to the test antigen and the procymidone standard drug under different pH conditions was evaluated.

Second, experimental results

The inhibition ratio can be calculated from the inhibition ratio formula of example 3. The determination result is shown in fig. 8, the binding activity of the nanobody NbFM5 with the amino acid sequence shown in SEQ ID No.1 to the detection antigen and the small molecule analyte is basically stable and unchanged within the range of pH 5.4 to 9.4, and outside this range, the binding activity of the antibody to the detection antigen and the small molecule analyte gradually decreases as the pH deviates from the neutral degree to a greater extent. When the pH was lowered to 2.4, the antibody was substantially inactivated, whereas when the pH was 10.4, the antibody inhibition rate was only reduced by about 30%. In general, NbFM5 can maintain better antigen binding activity over a wider pH range, but is more suitable for working in alkaline environments.

Example 12 storage stability determination of Nanobody NbFM5 at different temperature conditions

First, experiment method

0.03% preservative ProClin 300(v/v) and 0.01% protease inhibitor (v/v) are respectively added into a nano antibody NbFM5 solution with the amino acid sequence shown as SEQ ID NO.1, 20 mu L of each part is subpackaged, and the parts are respectively placed in four temperatures (-20, 4, 25 and 37 ℃) which are most common in daily use and are protected from light for storage. The kit of example 7 was used to determine the binding ability of the nanobody NbFM5 with the amino acid sequence shown in SEQ ID No.1 to the test antigen and the procymidone standard drug (3ng/mL) after storage for different time periods (0, 7, 14, 30, 60Day) at different temperatures, respectively.

Second, experimental results

The storage stability of the antibody after the preservative is added is proved, and the shelf life of the antibody is determined so as to have reference value in practical application. The inhibition rate can be calculated by the inhibition rate formula in the experimental method of example 3. As shown in fig. 9, after the nanobody NbFM5 with the amino acid sequence shown in SEQ ID No.1 is added with a preservative and a protease inhibitor, the binding activity of the nanobody NbFM5 with the antigen to be detected and the procymidone standard drug is substantially unchanged after being stored at different temperatures for one month, even when the storage time reaches two months, the binding activity of the nanobody NbFM5 antigen stored only at 37 ℃ is reduced to 41%, and the binding activity of the antibody stored at other temperatures is still substantially 100%. The nano antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 has high storage stability, and can be transported, stored and used for a long time at room temperature after being added with a common preservative agent.

It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and ideas, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> method and kit for detecting procymidone

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Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Glu Val Phe Thr Ser Lys Ile Ala Thr Gly

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Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val

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Ala Ile Thr His Thr Gly Ser Gly Asn Pro Trp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Leu Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Pro Glu Asp Thr Ala Thr Tyr Thr Cys

85 90 95

Ala Ala Arg Glu Ala Gly Ser Trp Glu Leu Arg Arg Leu Ser Pro Glu

100 105 110

Trp Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Val Phe

20 25

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Ala

1 5 10 15

Ile

<210> 4

<211> 38

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn

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Ala Lys Leu Thr Val Tyr Leu Gln Met Asn Asn Leu Arg Pro Glu Asp

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Thr Ala Thr Tyr Thr Cys

35

<210> 5

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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<400> 9

gaggtgcagc tgctggattc tgggggaggc tcggtacagg ctggagggtc tctgagactc 60

tcgtgtgaag tttttacaag caagatagca accggctaca tgggctggtt ccggcaggct 120

ccgggtaagg agcgcgaggc cgtcgcaatt actcataccg gtagtggaaa cccatggtat 180

gccgactccg tgaagggccg cttcaccatc tcccaagaca acgccaagct cacggtgtat 240

ctgcaaatga acaacctgag acctgaggac actgccacgt acacctgtgc ggcccgggag 300

gcagggtctt gggagctcag gcggttatct ccagaatggt atacctactg gggccagggg 360

accctggtca ccgtctcctc a 381

Claims

1. The nanometer antibody for specifically recognizing procymidone is characterized in that the amino acid sequence of the nanometer antibody is shown in SEQ ID NO. 1.

2. A gene for coding a specific recognition procymidone nano antibody is characterized in that the nucleotide sequence is shown as SEQ ID NO. 9.

3. A recombinant vector comprising the gene of claim 2 linked thereto.

4. A recombinant cell comprising the expression vector of claim 3 or capable of expressing the nanobody of claim 1.

5. The use of one or more of the nanobody of claim 1, the gene of claim 2, the recombinant vector of claim 3 and/or the recombinant cell of claim 4 in the detection of procymidone and/or in the preparation of a procymidone immunological detection kit.

6. A method for the non-diagnostic detection of procymidone, using nanobodies according to claim 1.

7. The detection method according to claim 6, wherein the detection is carried out based on an indirect ELISA method using a procymidone complete antigen obtained by coupling a procymidone hapten of formula (I) with a carrier protein as a detection antigen and the nanobody of claim 1 as a detection antibody,

8. a kit for detecting procymidone, which comprises the nanobody of claim 1.

9. The kit of claim 8, further comprising a procymidone complete antibody obtained by coupling the procymidone hapten and a carrier protein as a detection antigen, wherein the nanobody as claimed in claim 1 is used as a detection antibody,

10. the kit of claim 8, further comprising an enzyme-labeled secondary antibody, a color-developing agent, and a terminator.