CN114644712B

CN114644712B - Method and kit for detecting procymidone

Info

Publication number: CN114644712B
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: 2023-09-26
Anticipated expiration: 2041-12-23
Also published as: CN114644712A

Abstract

The invention discloses a method and a kit for detecting procymidone, which utilize a procymidone-resistant nano antibody with an amino acid sequence shown as SEQ ID NO.1, establish a method for detecting procymidone and develop a kit for detecting procymidone based on the method. The nano antibody prepared by the invention can be applied to the detection of the actual sample of procymidone residues, 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 nano antibodies of other small molecular substances, 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, low-cost, high-efficiency and broad-spectrum diformimine systemic fungicide, and has the action mechanism of inhibiting the synthesis of triglyceride in bacteria, has special effect on Botrytis and sclerotinia fungi, and is mainly used for preventing and treating gray mold, sclerotinia of fruit trees, vegetables and flowers, and black spot of other crops or foods such as pseudo-ginseng, ginseng and the like. Although procymidone is not high in toxicity, residues in fruits and vegetables can enter animals or human bodies through food chains, and metabolite dichloroaniline of procyanidine can generate certain toxicity to organisms, and can cause malformation, atrophy, lesions and the like of mammal sexual organs in severe cases. In recent years, because the procymidone is widely used on fruits and vegetables, the procymidone becomes one of pesticide varieties with higher pesticide residue detection rate and exceeding rate, especially the procymidone exceeding event in the Chinese chives is subjected to medium-frequency exposure in various supervision and spot inspection, so that the detection of procymidone pesticide residues is necessary to be enhanced.

Currently, most methods for detecting procymidone residues are gas chromatography, gas chromatography-mass spectrometry and liquid chromatography. Although the instrument method has 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 instrument and the like exist, and the instrument method can only be used for sampling detection of small batches of samples and cannot meet the requirements of on-site and rapid detection of large batches of samples.

The ELISA detection 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 commercial kit for on-site detection, and is widely applied to the detection of pesticide and veterinary drug residues and trace toxins. The key to success of the immunoassay method is to prepare antibodies with high sensitivity and high specificity, monoclonal antibodies and polyclonal antibodies are the most widely used at present, but the traditional antibodies have the defects of long development period, lower stability, harsh preservation conditions and the like, and the preparation of the stable antibodies with strong specificity and high sensitivity to procymidone has important significance for establishing a quick, sensitive and accurate procymidone detection method.

The nanometer antibody (Variable domain of heavy chain of heavy chain antibody, VHH) is a novel genetic engineering antibody, is derived from the variable region of heavy chain antibodies in camels and shark animals, and has the characteristics of low immunogenicity, high stability, high affinity, high specificity and the like. Nanobodies are easier to store and transport than conventional antibodies, and can refold and restore their antigen affinity efficiently even after denaturation under extreme conditions of high temperature, chemistry, and pressure. In addition, the nano antibody can be expressed in a large quantity through 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 overcome the defects and the shortcomings of the existing method for detecting procymidone, and provides a procymidone nano antibody, a preparation method and application thereof.

It is a first object of the present invention to provide a nanobody against procymidone.

It is a second object of the present invention to provide a gene encoding the nanobody.

A third object of the present invention is to provide a recombinant vector comprising the nucleotide sequence of the gene of the second object of the present invention.

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

A fifth object of the present invention is to provide the use of one or more of said nanobody, said gene, said recombinant vector, or said recombinant cell in an immunological detection assay for detecting procymidone.

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

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

In order to achieve the above object, the present invention is realized by the following means:

according to the invention, a Bactrian camel immune antibody library is constructed, a phage display technology is used, a detection antigen solid phase is coated on an ELISA plate, and the Bactrian camel immune antibody library is put into the Bactrian camel immune antibody library for affinity panning, so that a nano antibody capable of resisting specific binding of procymidone is obtained, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1. The method is applied to the immunological detection analysis of the procymidone, and a rapid, sensitive and stable procymidone detection method is established through the immunological detection analysis.

The invention therefore claims the following:

a nanometer antibody specifically recognizing procymidone has an amino acid sequence shown in SEQ ID NO. 1.

Wherein, the framework regions (FR 1-FR 4) 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 (CDR 1-CDR 3) are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO. 8.

A nucleotide sequence of the gene for encoding the nano antibody for specifically recognizing the procymidone is shown as SEQ ID NO. 9.

Specifically:

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

EVQLLDSGGGSVQAGGSLRLSCEVFTSKIATGYMGWFRQAPGKEREAVAITHTGSGNPWYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTCAAREAGSWELRRLSPEWYTYWGQGTLVTVSS；

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

EVQLLDSGGGSVQAGGSLRLSCEVF；

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

MGWFRQAPGKEREAVAI；

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

WYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTC；

the amino acid of the framework region FR4 of the nano antibody is shown as 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 nano antibody is shown in SEQ ID NO. 7:

THTGSGNP；

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

AAREAGSWELRRLSPEWYTY；

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

GAGGTGCAGCTGCTGGATTCTGGGGGAGGCTCGGTACAGGCTGGAGGGTCTCTGAGACTCTCGTGTGAAGTTTTTACAAGCAAGATAGCAACCGGCTACATGGGCTGGTTCCGGCAGGCTCCGGGTAAGGAGCGCGAGGCCGTCGCAATTACTCATACCGGTAGTGGAAACCCATGGTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCAAGACAACGCCAAGCTCACGGTGTATCTGCAAATGAACAACCTGAGACCTGAGGACACTGCCACGTACACCTGTGCGGCCCGGGAGGCAGGGTCTTGGGAGCTCAGGCGGTTATCTCCAGAATGGTATACCTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA。

a recombinant vector, which is connected with the coding gene.

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

The application of one or more of the nanobody, the gene, the recombinant vector and/or the recombinant cell in detecting procymidone and/or preparing 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 competition ELISA method, the complete antigen of procymidone obtained by coupling procymidone hapten and carrier protein as shown in formula (I) is used as detection antigen, the nano antibody is used as detection antibody,

preferably, the carrier protein is Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA) or chicken Ovalbumin (OVA).

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

more preferably, the sample to be detected and the nano antibody with the amino acid sequence shown as SEQ ID NO.1 are sequentially added into the solid phase carrier coated with the detection antigen, and after full reaction, liquid is discarded and washed; adding enzyme-labeled antibody, fully reacting, discarding liquid, washing, and discarding liquid and washing after full reaction; performing a color reaction, and terminating the reaction; OD values at 450nm were read.

In a specific embodiment, the TMB developing solution, in which developing solution A is mixed with developing solution B, undergoes a developing reaction of 10% H ₂ SO ₄ (v/v) terminate the reaction.

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

Preferably, the kit also comprises a complete procymidone antigen obtained by coupling the procymidone hapten and carrier protein of the formula (I), the nano antibody is used as a detection antibody,

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

Preferably, the kit also comprises enzyme-labeled secondary antibodies, a color developing agent and a terminator.

More preferably, the color developer is color developing solution a and color developing solution B.

More preferably, the terminator is 10% H ₂ SO ₄ (v/v)。

More preferably, the second enzyme-labeled antibody is an Anti-VHH-HRP 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 quantity by means of genetic engineering recombinant expression. The recombinant expression mode of gene engineering is to clone the gene encoding the nanometer antibody into expression vector and to prepare the nanometer antibody in protein expression mode. After the nano antibody is expressed by prokaryote, immunological detection analysis is carried out in the form of protein.

The nano antibody prepared by the invention can be applied to the detection of the actual sample 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 nano antibodies of other small molecular substances, and has higher application value.

Drawings

FIG. 1 is a chart of ultraviolet scanning spectra of procymidone hapten, carrier protein and complete antigen.

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

FIG. 3 is a SDS-PAGE of Pythiaside 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 graph showing the activity of nanobody NbFM5 in methanol/acetonitrile/acetone at various concentrations.

FIG. 7 is a graph showing the activity of NbFM5 nanobody after incubation at 70℃for various times.

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

FIG. 9 is a graph showing the activity of NbFM5 nanobody stored at different temperatures for different times.

Detailed Description

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

EXAMPLE 1 preparation of the immune antigen PRM-LF and detection antigen PRM-BSA

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

1. experimental method

1. Preparation of complete antigen PRM-LF

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

the specific operation method is as follows: 2.876mg of EDC and 1.725mg of NHS are weighed out and 0.1mL of DMF is added to dissolve the solid. 3.518mg of procymidone hapten is added into the mixture solution, and the mixture solution is stirred or oscillated at room temperature for 4 hours to activate the hapten. 20mg 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. The activated procymidone hapten is dropwise added dropwise in an ice bath under stirring, and the solution is determined to be in an alkaline range by using pH test paper and stirred at room temperature overnight. Dialyzing with PBS for 3 times, and freezing at-20deg.C. Scanning the coupling raw material (hapten and carrier protein LF) and the coupling product by an ultraviolet spectrophotometry, and judging whether the coupling is successful or not by utilizing an ultraviolet absorption spectrum obtained by scanning.

2. Preparation of complete antigen PRM-BSA

The procymidone hapten with the structural formula shown as (I) and Bovine Serum Albumin (BSA) are coupled by an active ester method to form an artificial detection antigen, the structural formula of which is shown as (III),

the specific operation method is as follows: 6.535mg of EDC and 3.92mg of NHS were weighed out and 0.15mL of DMF was added to dissolve the solid. 7.995mg of procymidone hapten is added into the mixture solution, and the mixture solution is stirred or oscillated at room temperature 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. The activated hapten is dropwise added dropwise in an ice bath under stirring, and the solution is determined to be in an alkaline range by using pH test paper and stirred at room temperature overnight. Dialyzing with PBS for 3 times, packaging, and freezing at-20deg.C. Dialyzing with PBS for 3 times, packaging, and freezing at-20deg.C. Scanning the coupling raw material (hapten and carrier protein BSA) and the coupling product by an ultraviolet spectrophotometry, and judging whether the coupling is successful or not by utilizing the ultraviolet absorption spectrum obtained by scanning.

2. Experimental results

The UV scan spectra of procymidone hapten, carrier protein (LF, BSA) and conjugate product are shown in FIG. 1. The characteristic peaks of procymidone hapten are at 210nm and 280nm, LF ultraviolet absorption peak is at 210nm and 280nm, and BSA ultraviolet absorption peak is at 228nm and 280 nm. The ultraviolet absorption peaks of the complete antigen PRM-LF II after coupling the carrier protein LF and the procymidone hapten are 229nm and 280nm, and the ultraviolet absorption peaks of the complete antigen PRM-BSA III after coupling the carrier protein BSA and the procymidone hapten are 242nm and 278nm. Therefore, the ultraviolet absorption peaks of the two artificial antigens are basically and obviously shifted from those of the hapten and the carrier protein, and the absorbance is also changed, which indicates that the coupling of the procymidone hapten with the carrier protein LF and BSA is successfully realized.

EXAMPLE 2 construction of Bactrian camel immune antibody library

1. Bactrian camel immunization protocol

Animals were immunized with healthy Bactrian camels and the complete antigen PRM-LF of formula (II) prepared in example 1 was used as the immunizing antigen and subcutaneously injected into the neck of the humpback at a dose of 0.5mg of immunizing antigen each time. The first immunization was performed by emulsifying 0.5mL of complete Freund's adjuvant with the immunizing antigen, and the subsequent booster immunization was performed by emulsifying 0.5mL of incomplete Freund's adjuvant with the antigen, and 4 booster immunizations were performed every 2 weeks. After the fourth and fifth immunization, 50-100 mL of peripheral blood is collected and used for constructing a nano antibody library.

2. Isolation of Bactrian camel lymphocytes

The method for collecting the peripheral blood of the Bactrian camel needs to separate lymphocytes as soon as possible, and comprises the following specific operation methods: the peripheral blood of Bactrian camel was diluted by mixing with sterile physiological saline in equal volume in a clean container without rnase. The diluted peripheral blood is centrifuged by commercial lymphocyte separating medium, and the lymphocyte separating medium is distributed to different depths after the blood cells with different densities are centrifuged, wherein the lymphocyte is positioned at the depth of about 1/3 of the liquid surface to form a white cell layer. At room temperature, 850g was centrifuged for 25min. Carefully collecting the cell sediment in a clean container without RNase by using a rubber head dropper or a pipette, washing redundant lymphocyte separating liquid on the surface of the cell by using sterile physiological saline, and centrifuging. Adding TRNSol as lysate into the collected lymphocyte, repeatedly blowing with RNase-free pipette or rubber head dropper until the lymphocyte is completely lysed in TRNSol, and storing at-80deg.C for use.

3. Extraction of Total RNA

And 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 specification of the RNA extraction kit. After total RNA extraction, a few samples were taken for nucleic acid electrophoresis and RNA concentration was determined in an ultra-micro spectrophotometer (nanodrop).

4. cDNA Synthesis

And (3) reversely transcribing mRNA in the RNA sample obtained in the previous step into cDNA by using a commercial reverse transcription kit, and performing operation according to the instruction. After the reverse transcription is completed, the products are uniformly mixed, and then are subpackaged in different sterile centrifuge tubes and stored in an environment of-80 ℃.

5. Amplification of nanobody target gene VHH

The target gene VHH of the nano antibody is obtained by adopting a nested PCR (polymerase chain reaction) two-step method for amplification, and the sequence of the used primer is shown in the table 1:

TABLE 1 heavy chain antibody Gene primer sequences of Bactrian camels

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

TABLE 2 first-step reaction system of nested PCR and reaction conditions

The first step PCR product showed two product bands of 1000bp and 750bp after nucleic acid electrophoresis, and the 750bp band was recovered by gel cutting and the concentration was measured.

Second round PCR the second round PCR amplification was performed using the recovered product of the first round PCR as 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) Cleavage of the target Gene VHH and vector pComb3xss

And (3) carrying out enzyme digestion reaction on the VHH target gene and the pComb3xss vector by adopting Sfi I enzyme. Enzyme cutting conditions: the reaction is carried out for 16h at a constant temperature of 50 ℃.

The enzyme cutting product of the pComb3xss vector is used for recovering a band with the molecular weight of 3500bp through agarose gel; the VHH gene cleavage products are directly cleaned and recovered by a DNA recovery kit.

(2) Ligation of cleavage products

The vector pComb3xss and VHH fragment were mixed uniformly (molar ratio 1:3), reacted at 16℃for 16 hours, and then recovered by cleaning with a DNA recovery kit.

(3) Shock conversion

mu.L of the ligation product was added to 50. Mu.L of electrotransformation competent E.coil TG1, and after gentle mixing, the mixture was transferred to a 0.1cm electrotransformation cup for electric shock transformation (voltage: 1.8 kv), and immediately after electric shock, 950. Mu.L of SOC medium preheated to 37℃was added to the electrotransformation cup, and shaking bacteria at 250rpm for 1 hour at 37℃for cell recovery.

100. Mu.L of resuscitating bacteria liquid is taken for gradient dilution, 100. Mu.L of each concentration gradient diluted bacteria liquid is coated on an LB-Amp culture dish with the diameter of 90mm as a counting plate, and the culture is carried out at 37 ℃ overnight. The residual undiluted resuscitated bacterial solution is coated on LB-Amp culture dishes with the diameter of 120mm, each 1mL of bacterial solution is coated on 2-3 culture dishes to serve as amplification plates, and amplification culture is carried out at 37 ℃ overnight.

Counting the bacterial colony number on the culture dish, calculating the total bacterial number in the resuscitating bacterial liquid, performing multiple electric shock conversion to ensure that the total bacterial colony number reaches 10 ⁷ Above cfu, the number is the library capacity of the nanobody gene library.

Scraping the colony of the transgenic escherichia coli 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, carrying out gradient dilution to determine the cell number, and freezing the rest bacterial liquid at-80 ℃ after subpackaging, thus obtaining the procymidone nanobody gene library.

7. Phage rescue

Inoculating cells with a 10-fold reservoir capacity or more into 200mL LB-Amp according to the measurement result of the transgenic escherichia coli cell number, and culturing at 37 ℃ at 250rpm until the logarithmic phase; 1mL of the mixture was added to give a titer of 10 ¹² The helper phage M13K07 above cfu/mL was incubated at 37℃for 30min at 250rpm for 1h, kanamycin (70. Mu.g/mL) was added, and incubated at 37℃at 250rpm overnight. Centrifuging at 12000rpm for 15min at 4 ℃, taking supernatant, adding 1/4 volume of PEG/NaCl solution (100 g of PEG 8000 and 73.05g of sodium chloride are added with water to fix the volume to 500 mL), and carrying out ice bath for 2-3 h. Centrifuge at 12000rpm 4℃for 15min, discard supernatant, resuspend pellet with 1mL TBS, transfer to 2mL centrifuge tube, centrifuge at 12000rpm 4℃for 5min, and filter with 0.22 μm polyethersulfone. Measurement of 10. Mu.L of phage And (3) determining titer, adding glycerol with the final concentration of 50%, and preserving at-80 ℃ to obtain the procymidone nanobody phage library.

Example 3 affinity panning and identification of nanobodies

1. Experimental method

1. Affinity panning of nanobodies

(1) Immobilization of detection antigen

With coating liquid (0.375 g Na ₂ CO ₃ And 0.7325g NaHCO ₃ Adding water to a volume of 250 mL), diluting the complete antigen PRM-BSA with the structural formula shown in (III) prepared in example 1 to 1 mug/mL, adding the diluted complete antigen to the microwells of an ELISA plate, and standing at 4 ℃ for overnight at 100 mug/well. The next 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. Pouring out the liquid in the hole, beating the liquid on absorbent paper, drying the liquid at 37 ℃ for 1h, and storing the liquid at 4 ℃ for standby.

(2) Positive phage selection

BSA was added to the above-mentioned Pythium nano-antibody phage library to give a final BSA concentration of 1% (w/v), phage was added to 3 microwells with immobilized antigen, 100. Mu.L of phage was added to each well, and incubated at 37℃for 1 hour. The phage released from the wells were discarded, the wells were washed 10 times with PBST and 5 times with PBS.

Pythium standard substance with the concentration of 5 mug/mL is added into each hole, 100 mug/mL of each hole is subjected to shake incubation for 4 hours under the condition of 37 degrees to perform competition reaction, and liquid in each micropore is collected into a sterile centrifuge tube. The phage at this point is called "competing output". To prevent loss of diversity, trypsin Trypsin (10 mg/mL) was added to the wells at the end of the competition elution for 30min, and the liquid in the microwells was collected into sterile centrifuge tubes, called "enzyme wash output", and the first round of screening was completed.

Phage titer was measured before and after screening, and the specific procedure was as follows:

taking 10 μL of two output phages, respectively performing 10-fold gradient dilution, selecting desired titer and two dilutions adjacent to each other (generally input dilution of 10) ⁷ 、10 ⁸ 、10 ⁹ Output dilution factor of 10 ² 、10 ³ 、10 ⁴ ) mu.L of dilution was used to infect 100. Mu.L of log phase E.coil TG1, and the mixture was allowed to stand at 37℃for 30min, and LB-Agar-Amp plates were applied, and after overnight incubation, colony counts were performed and phage titers were calculated as follows:

(3) Phage amplification

The phage amplified by the screening is used for the next screening, and the phage amplification method is as follows:

half of the competition eluting output phage was added to 4mL log phase e.coil TG1 and allowed to stand for 30min. LB medium was added to a total volume of 10mL and Amp was added to a working concentration (10. Mu.g/mL), shaking was performed at 37℃and 250rpm for 30min, and 50. Mu.L of the medium was added to a titer of 10 ¹² cfu/mL of M13KO7 helper phage was allowed to stand for 30min, shaken at 37℃and 250rpm for 1h, LB was added to a total volume of 100mL, amp and Kana were added, and shaking was performed at 37℃and 250rpm overnight.

The bacterial solution is centrifuged, the supernatant is transferred to a new centrifuge bottle, 1/4 volume of a mixed solution of PEG and NaCl (100 g PEG 8000 and 73.05g sodium chloride are added with water to a volume of 500 mL), the mixture is stirred evenly, the mixture is centrifuged for 20min at 12000rpm for 2h in an ice bath, the supernatant is discarded and the residual moisture is removed as much as possible by back-off on absorbent paper, the white precipitate is resuspended by 1mL TBS containing 1% BSA (w/v), and the mixture is stored at-20 ℃ after passing through a 0.22 mu m polyethersulfone filter membrane, and the mixture is used as the next round of screening "input", and the screening scheme of the step (2) is repeated.

The screening steps are carried out for 4 times, the concentration of the detected antigen is fixed to be 1ug/mL, and the concentration of the medicine used in the competition reactions of the 2 nd round, the 3 rd round and the 4 th round is respectively reduced to 2000ng/mL,400ng/mL and 40ng/mL.

2. Identification of Positive clones

And (3) identifying positive phage clones by adopting an indirect enzyme-linked immunosorbent assay. The specific method comprises the following steps:

(1) Immobilization of antigen

Coating liquid for detecting antigen (0.375 g Na) ₂ CO ₃ And 0.73 of25g NaHCO ₃ Water was added to volume 250 mL) and diluted to 1 μg/mL, and left to stand overnight at 4 ℃. The next day, after washing twice with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20), 2% skimmed milk powder (w/v) was prepared with PBS, 150. Mu.L was added to each well, the wells were blocked for 2h at 37℃and the blocked solution was discarded, and the wells were dried by flash and baked at 37℃for 60min and stored at 4℃for use.

(2) Nanobody miniexpression

The third and fourth rounds of elutriation were performed by randomly selecting 96 single colonies on the output titer assay plates for competition elution, inoculating into 96 well plates each containing 0.5mL LB-Amp, and simultaneously inoculating one E.coil TG1 single colony not infected with phage as a negative control, and incubating overnight at 37℃to serve as a bacterial liquid "master".

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

(3) Identification of positive clones by ELISA

Centrifuging the deep pore plate at 4000rpm for 20min, taking two ELISA plates with immobilized antigens, wherein plate 1 is used as a titer plate for detecting antibodies, and 50 mu L of PBS is added into each pore; plate 2 was used as a inhibition plate, PBS containing 50ng/mL procymidone standard drug was added, and supernatant was aspirated from the centrifuged 96-well plate, and 50. Mu.L of each well was added to the corresponding numbered plate well. Incubation was carried out at 37℃for 30min, washed five times with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20), and the liquid in the wells was dried. Anti-VHH-HRP secondary antibody (GenScript, A01861-200) was diluted 5000-fold with PBST, 100. Mu.L was added to each well and incubated at 37℃for 30min. Washing with 20-fold diluted washing solution PBST (0.01M PBS,0.05%Tween-20) for five times, drying the liquid in the wells, adding 100 μl of TMB color development solution (Solarbio, PR 1210) which is prepared by mixing color development solution A and color development solution B in equal volumes in advance into each well, and developing at 37deg.C for 10min; 50. Mu.L of stop solution 10% H was added ₂ SO ₄ (v/v) terminating the reaction; the absorbance at 450nm was measured with a microplate reader.

The inhibition rate of each positive clone can be calculated according to the following formula. Clones with OD values 3 times greater than that of the negative control wells in plate 1 and with significant inhibition (inhibition > 20%) were selected, the numbers of their corresponding wells were recorded, and the bacterial solutions of the corresponding wells in the master plate were transferred to a sterile centrifuge tube and added to glycerol for cryopreservation.

2. Experimental results

And (3) sending the strain of the nanobody obtained through indirect competition ELISA identification to a sequencing company for gene sequencing, and obtaining the amino acid sequence of the nanobody according to a DNA sequencing result and a codon table. The results show that 7 Pythium nano-antibodies with different sequences are obtained and are respectively named as NbFM 1-NbFM 7. As shown in FIG. 2, the inhibition rates of NbFM1 to NbFM7 were 78.91%, 74.68%, 88.61%, 44.01%, 93.57%, 38.02% and 36.19%, respectively, wherein NbFM5 showed the most prominent inhibition rate in the indirect competition ELISA assay.

NbFM5 has an amino acid sequence shown as SEQ ID NO.1, the nano-antibody comprises 4 FR framework regions and 3 CDR complementarity determining regions, the arrangement sequence is FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, wherein the framework regions (FR 1-FR 4) are sequentially shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SE1% Q ID NO. 5; the complementarity determining regions (CDR 1-CDR 3) 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 obtained gene for encoding the nano NbFM5 is shown as SEQ ID NO. 9.

Specifically:

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

EVQLLDSGGGSVQAGGSLRLSCEVF；

MGWFRQAPGKEREAVAI；

WYADSVKGRFTISQDNAKLTVYLQMNNLRPEDTATYTC；

WGQGTLVTVSS；

TSKIATGY；

THTGSGNP；

AAREAGSWELRRLSPEWYTY；

EXAMPLE 4 Mass production of nanobody NbFM5

The strain plasmid carrying the expression vector of NbFM5 gene (the nucleotide sequence is shown as SEQ ID NO. 9) is extracted by a kit, transformed into competent E.coil BL21 (DE 3) and coated on LB-Agar-Amp plate culture to obtain single colony. A single colony was inoculated into 10mL of LB-Amp, and cultured overnight at 37℃and 250 rpm. The overnight cultures were inoculated at a 1% (v/v) ratio into 100mL LB-Amp, incubated at 37℃at 250rpm to logarithmic phase, IPTG was added to a working concentration of 1mM, incubated at 37℃at 250rpm overnight.

Centrifuging bacterial liquid, discarding supernatant, and extracting antibody Zhou Zhiqiang protein by a sucrose osmotic freeze thawing method, wherein the specific method comprises the following steps: adding 3.75mL of TES solution to resuspend and precipitate, freezing for more than 2 hours at the temperature of minus 80 ℃, adding 15mL of TES diluted by 5 times after melting in water bath at the temperature of 37 ℃, shaking for 2 hours at 250rpm to fully extract protein, centrifuging to leave supernatant, purifying the supernatant by nickel-agarose gel affinity chromatography to obtain the nano antibody NbFM5 shown in the figure 3 (the amino acid sequence is shown as SEQ ID NO. 1).

EXAMPLE 5 determination of working concentration and sensitivity of nanobody NbFM5

1. Experimental method

1. Immobilization of antigen

With coating liquid (0.375 g Na ₂ CO ₃ And 0.7325g NaHCO ₃ Adding water to a volume of 250 mL), diluting the complete antigen PRM-BSA with the structural formula shown as (III) prepared in the example 1 to a series of concentrations of 2000, 1000, 500, 250ng/mL and the like, respectively adding the complete antigen PRM-BSA into the microwells of an ELISA plate, and standing at 4 ℃ for overnight at 100 mu L of each microwell. The next 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. Pouring out the liquid in the hole, beating the liquid on absorbent paper, drying the liquid at 37 ℃ for 1h, and storing the liquid at 4 ℃ for standby.

2. Determination of antibody working concentration

And diluting the purified nano antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 by PBS (phosphate buffer solution) to respectively dilute the nano antibody NbFM5 into a series of working solutions with different concentrations. 50 μl PBS per well was added to the ELISA plates at different coating concentrations, followed by 50 μl gradient diluted antibodies, and 3 well replicates were performed for each antibody concentration, with 3 wells for blank control (100 μl PBS). The wells were washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) after incubation at 37℃for 30min, 100. Mu.L of Anti-VHH-HRP secondary antibody (GenScript, A01861-200) diluted 5000-fold with PBST was added to each well after drying on absorbent paper. Incubating at 37deg.C for 30min, washing microwells with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) for 5 times, drying on absorbent paper, adding 100 μL TMB color development solution (Solarbio, PR 1210) pre-mixed with equal volumes of color development solution A and color development solution B into each well, incubating at 37deg.C in the absence of light Incubating for 10min, adding 50 μl of stop solution 10% H into each well ₂ SO ₄ (v/v) the OD at 450nm was read on a microplate reader.

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

3. Drawing standard curve by indirect competition ELISA

The procymidone standard was diluted with PBS to a series of procymidone solutions of different concentrations, 50 μl of procymidone of different concentrations was added to each well of the elisa plate of different coating concentrations, 3-well replicates were performed for each concentration, and a 3-well drug blank (50 μl PBS) was prepared. The nanometer antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 is diluted to working concentrations of 43.33, 43.33, 43.33 and 54.17ng/mL respectively by PBS, and 50 mu L of diluted antibody is added into the corresponding holes with different coating concentrations. The wells were washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) after incubation at 37℃for 30min, 100. Mu.L of Anti-VHH-HRP secondary antibody (GenScript, A01861-200) diluted 5000-fold with PBST was added to each well after drying on absorbent paper. Incubating at 37deg.C for 30min, washing microwells with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) for 5 times, beating to dry on absorbent paper, adding 100 μL of TMB color development solution (Solarbio, PR 1210) pre-mixed with equal volumes of color development solution A and color development solution B into each well, incubating at 37deg.C in the absence of light for 10min, adding 50 μL of stop solution 10% H into each well ₂ SO ₄ (v/v) the OD at 450nm was read on a microplate reader.

Average value of OD450nm value of drug blank group was recorded as B ₀ OD450nm average values at different drug concentrations were recorded as B _x B was calculated with Excel at different drug concentrations _x /B ₀ Ratio and standard deviation of each set of parallel data. On the abscissa of drug concentration, B _x /B ₀ And drawing a scatter diagram in Origin software and performing logistic function fitting to establish an indirect competition standard curve by taking the ratio as an ordinate. In addition, the obtained standard curve parameters IC50 and B with different coating concentrations ₀ 、B ₀ IC50 ProcessThe selected optimal antigen-antibody working concentrations are compared.

2. Experimental results

The antigen-antibody working concentration of the nano antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 is optimized by a chessboard titration method. As a result, as shown in FIG. 4, the IC50 value gradually decreased with decreasing concentration of the antigen to be detected, B ₀ First drop of IC50 value, relatively low IC50 value and B at a coating concentration of 500ng/mL ₀ The IC50 was maximal, 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 antigen-antibody concentration selected above, an indirect competition ELISA standard curve established based on the nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 is shown in FIG. 5, and it can be seen that the standard curve is S-shaped, has good linear correlation, the detection range is 0.19-2.38 ng/mL, the IC50 is 0.67ng/mL, the minimum detection Limit (LOD) is 0.13ng/mL, and the detection sensitivity is high.

Example 6A method for detecting Pythium

1. Pre-coating plate: with coating liquid (0.375 g Na ₂ CO ₃ And 0.7325g NaHCO ₃ Adding water to a volume of 250 mL), 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 to the microwells of an ELISA plate, and standing at 4 ℃ for overnight at 100 mu L of the microwells. The next 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. Pouring out the liquid in the hole, beating the liquid on absorbent paper, drying the liquid at 37 ℃ for 1h, and storing the liquid at 4 ℃ for standby.

2. Blank hole B for marking standard substance ₀ Wells, standards and samples, 3-well replicates were performed. At B ₀ To each well was added 50. Mu.L of the standard solution at different concentrations and 50. Mu.L of the diluted sample solution. 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 the diluted antibody solution was added to all the above wells, and incubated at 37℃for 30min.

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

5. mu.L of TMB color solution (Solarbio, PR 1210) previously mixed with an equal volume of color solution A and color solution B was added to each well and incubated at 37℃for 10min in the absence of light.

6. mu.L of stop solution 10% H was added to each well ₂ SO ₄ (v/v) the OD at 450nm was read on a microplate reader.

3. Interpretation of results

Average value of standard blank OD450nm value is recorded as B ₀ The average value of OD450nm and sample holes to be measured under different drug concentrations is recorded as B _x Calculating B of different drug concentrations or sample wells _x /B ₀ Ratio and standard deviation of each set of parallel data. On the abscissa of the logarithmic value of the standard substance concentration, B _x /B ₀ The 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 can be obtained from the curve, namely the logarithmic value of the procymidone concentration, and the anti-logarithmic value is the procymidone concentration 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 ratio.

Example 7A kit for detecting Pythium

1. Composition of the kit

1. Removable ELISA plates pre-coated with BSA coupled to procymidone antigen: with coating liquid (0.375 g Na ₂ CO ₃ And 0.7325g NaHCO ₃ Adding water to a volume of 250 mL), 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 to the microwells of an ELISA plate, and standing at 4 ℃ for overnight at 100 mu L of the microwells. The next 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 1 hour. Pouring out of the hole The liquid was patted dry on absorbent paper and baked at 37 ℃ for 1h.

2. Enzyme-labeled reagent: anti-VHH-HRP secondary antibody needs to be diluted 5000 times when in use.

3. Standard substance: pythium standard solutions of different concentrations.

4. Dilution of antibody with sample: PBS.

5. Dilutions of labeled antibodies: PBST.

6. Color development liquid A and color development liquid B.

7. Stop solution: 10% (v/v) H ₂ SO ₄ 。

8. Concentrated washes (20×): PBST (0.01M PBS,0.05%Tween-20) for washing the plate.

9. Detection of antibodies: the amino acid sequence of NbFM5 is shown in SEQ ID NO. 1.

2. Use of a kit

Numbering in advance, and marking blank holes B of standard substance ₀ Wells, standards and samples, 3-well replicates were performed. At B ₀ To each well was added 50. Mu.L of PBS, 50. Mu.L of different concentrations of standard solution was added to each well, and 50. Mu.L of diluted sample solution was added to each well. 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 the diluted antibody solution was added to all the above wells, and incubated at 37℃for 30min. The microwells were washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) and 100. Mu.L of enzyme-labeled secondary antibody was added to each well after drying on absorbent paper. Incubating at 37 ℃ for 30min, washing the microwells 5 times by using a washing solution PBST (0.01M PBS,0.05%Tween-20) diluted by 20 times, taking a dry on absorbent paper, adding 100 mu L of TMB developing solution (Solarbio, PR 1210) which is prepared by mixing the developing solution A and the developing solution B in equal volumes in advance into each well, incubating at 37 ℃ in a dark place for 10min, adding 50 mu L of stop solution into each well, and reading the OD value of 450nm on an enzyme label instrument within 5 min.

3. Interpretation of results

Average value of standard blank OD450nm value is recorded as B ₀ The average value of OD450nm and sample holes to be measured under different drug concentrations is recorded as B _x Different drugs were calculated using ExcelConcentration of substance or B of sample well _x /B ₀ Ratio and standard deviation of each set of parallel data. On the abscissa of the logarithmic value of the standard substance concentration, B _x /B ₀ The 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 can be obtained from the curve, namely the logarithmic value of the procymidone concentration, and the anti-logarithmic value is the procymidone concentration 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 ratio.

EXAMPLE 8 specific assay of nanobody NbFM5

1. Experimental method

The complete antigen PRM-BSA with the structural formula shown in the (III) prepared in the example 1 is taken as a detection antigen, the nano antibody NbFM5 is taken as a detection antibody, other 9 procymidone analogues (ethephon, dimethachlon, iprodione, folpet, dripping nasal discharge and hexahexa) and procymidone metabolite (3, 5-dichloroaniline) standard substance solutions are prepared, the kit of the example 7 is used, and standard curves of each procymidone analogue and metabolite are drawn according to the indirect competition ELISA method in the example 5, so that respective IC50 values are obtained. The cross-reactivity of each drug with nanobody NbFM5 was calculated using the following formula:

2. Experimental results

The results show that the cross reaction rate of the nanobody NbFM5 and procymidone metabolite 3, 5-dichloroaniline and analogues thereof such as ethephon, dimethachlon, iprodione, folpet, dripping nasal discharge, ethidium bromide and six-six is lower than 0.1 percent (IC 50 is more than 700 ng/mL); the nano antibody NbFM5 can specifically identify the procymidone, and the detection method established by the invention has high specificity for detecting the procymidone.

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

1. Experimental method

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

The binding capacities of the antibodies to the detection antigen and to the procymidone standard drug (3 ng/mL) were measured under different organic solvent conditions using the detection kit of example 7, and the tolerance of the nanobody NbFM5 having the amino acid sequence shown in SEQ ID NO.1 to different organic solvents was evaluated.

2. Experimental results

The above antibodies were diluted with PBS, so the actual organic concentrations to which the antibodies were exposed were 0%, 10%, 20%, 30%, 40%, 50%, respectively. The inhibition ratio can be calculated from the inhibition ratio formula of example 3. As shown in FIG. 6, the binding activity of NbFM5 nano antibody with the amino acid sequence shown in SEQ ID NO.1 with detection antigen and small molecule in low concentration methanol solution below 10% is less affected, and when the concentration of acetonitrile and acetone solution reaches 10%, the binding activity of the antibody with detection antigen can only be preserved by 30% and 40%. In addition, the binding activity of NbFM5 and detection antigen and small molecule drugs is drastically reduced with the increase of the concentration of the organic solvent. When the proportion of methanol, acetonitrile and acetone in the organic solvent mixed solution reaches 40%, 20% and 20% respectively, the antibody is completely deactivated.

The nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 has poor tolerance to three common organic solvents (methanol, acetonitrile and acetone) in pretreatment of a standard method, and the organic solvents should be removed in an antibody working solution as much as possible, so that the influence of the high-concentration organic solvents on the sensitivity of the antibody is avoided.

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

1. Experimental method

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

2. Experimental results

The inhibition rate can be calculated from the inhibition rate formula in the experimental method of example 3. The measurement result is shown in fig. 7, the binding activity of the nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 and the detection antigen and the binding activity of the small molecule to-be-detected object show a decreasing trend along with the extension of the incubation time, wherein the decreasing trend of the binding activity of the nano antibody and the small molecule to-be-detected object is more gentle. The nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 still has 50% antigen binding activity and 66% inhibition rate even after being heated for 1 hour at 70 ℃. The nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 can refold and restore most of antigen binding capacity after high-temperature denaturation, and the characteristic can lead the nano antibody NbFM5 to have great application advantages in a relatively high-temperature detection environment.

EXAMPLE 11 determination of the Activity of the nanobody NbFM5 at different pH conditions

1. Experimental method

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

2. Experimental results

The inhibition ratio can be calculated from the inhibition ratio formula of example 3. As shown in FIG. 8, the binding activity of the nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 with the detection antigen and the small molecule to-be-detected object is basically stable and unchanged within the pH range of 5.4-9.4, and the binding activity of the antibody with the detection antigen and the small molecule to-be-detected object is gradually reduced along with the increasing degree of pH deviation from neutrality outside the range. When the pH was lowered to 2.4, the antibody was substantially inactivated, whereas when the pH reached 10.4, the antibody inhibition was reduced by only 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 determination of storage stability of nanobody NbFM5 under different temperature conditions

1. Experimental method

Adding 0.03% preservative ProClin 300 (v/v) and 0.01% protease inhibitor (v/v) into NbFM5 solution of nano antibody with amino acid sequence shown in SEQ ID NO.1, respectively, subpackaging 20 μl each, and storing at four temperatures (-20, 4, 25, 37 ℃) which are most common in daily use in dark. The binding ability of the nanobody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 to the antigen to be detected (3 ng/mL) was determined by using the kit of example 7 after storage for different times (0, 7, 14, 30, 60 Day) under different temperature conditions.

2. Experimental results

The preservation of commercial antibodies generally requires the addition of a preservative or protease inhibitor, and this example demonstrates the storage stability of the antibodies after the addition of the preservative, and determines their shelf life so as to have a more reference value in practical applications. The inhibition rate can be calculated from the inhibition rate formula in the experimental method of example 3. As shown in FIG. 9, after the preservative and the protease inhibitor are added, the nano antibody NbFM5 with the amino acid sequence shown in SEQ ID NO.1 is stored for one month at different temperatures, the binding activity of the nano antibody NbFM5 with the amino acid sequence with the detection antigen and the procymidone standard drug is basically unchanged, even when the storage time reaches two months, the binding activity of the nano antibody NbFM5 stored at 37 ℃ is reduced to 41%, and the binding activity of the nano antibody stored at other temperature is basically 100%. The nano antibody NbFM5 with the amino acid sequence shown as SEQ ID NO.1 has high storage stability, and can be transported, stored and used for a long time under the room temperature condition after being added with a common preservative reagent.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Sequence listing

<110> agricultural university of south China

<120> method and kit for detecting procymidone

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

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 Thr Ser Lys Ile Ala Thr Gly

20 25 30

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

35 40 45

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

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

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

20 25 30

Thr Ala Thr Tyr Thr Cys

35

<210> 5

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Thr Ser Lys Ile Ala Thr Gly Tyr

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Thr His Thr Gly Ser Gly Asn Pro

1 5

<210> 8

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Trp Tyr Thr Tyr

20

<210> 9

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<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. A nano antibody for specifically recognizing procymidone is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

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

3. A recombinant vector, wherein the recombinant vector is linked to the gene of claim 2.

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

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

6. Use of one or more of the genes of claim 2, the recombinant vector of claim 3 and the recombinant cells of claim 4 for the detection of procymidone for non-diagnostic purposes.

7. A method for the non-diagnostic detection of procymidone, wherein the method uses the nanobody of claim 1.

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

9. A kit for detecting procymidone, comprising the nanobody of claim 1.

10. The kit according to claim 9, further comprising a complete antigen of procymidone obtained by coupling the hapten of procymidone with a carrier protein of formula (I), and a nanobody according to claim 1,

11. the kit of claim 10, further comprising an enzyme-labeled secondary antibody, a chromogenic agent, and a terminator.