CN114409795A

CN114409795A - Nano antibody for detecting diazinon and application thereof

Info

Publication number: CN114409795A
Application number: CN202111604801.5A
Authority: CN
Inventors: 徐振林; 黄丹彤; 何晓婷; 陈子键; 王弘; 沈玉栋; 雷红涛; 罗林
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-04-29
Anticipated expiration: 2041-12-24
Also published as: CN114409795B

Abstract

The invention discloses a nano antibody for detecting diazinon and application thereof, wherein the amino acid sequence of the nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence of a gene encoding the nano antibody is shown as SEQ ID NO. 9. The invention discloses a nano antibody which can be applied to the actual sample detection of diazinon residues. The nano antibody has the advantages of strong specificity, high temperature resistance, acid and alkali resistance, easiness in storage, low production cost, short production period and the like, can be used as a novel material for detecting diazinon, and has a good application prospect and a wide development space in the rapid and effective detection of the diazinon.

Description

Nano antibody for detecting diazinon and application thereof

Technical Field

The invention relates to the technical field of detection of pesticides, and particularly relates to a diazinon nano antibody and application thereof.

Background

Diazinon (dazin, DAZ) is a broad-spectrum and efficient organophosphorus insecticide, has contact poisoning, stomach poisoning, fumigating and certain systemic effects, and is used for preventing and treating ectoparasite diseases such as mites, flies, lice and ticks of livestock and various pests of important crops such as rice, cotton, vegetables and flowers. However, the diazinon is widely remained in environmental media such as water, soil and the like and food due to the large use of the diazinon and the limitation of application technology, and enters various organisms and human bodies through food chains. Diazinon is insecticidal by inhibiting the biological activity of acetylcholinesterase in insects, and accumulation of the neurotransmitter acetylcholine in other organisms can lead to a series of syndromes including anorexia, diarrhea, general weakness, muscle tremor, abnormal posture and behavior, depression and death. Therefore, strict maximum residual limit of diazinon residues in food is set all over the world, for example, the maximum residual limit of diazinon in food in China is 0.01-0.5 mg/kg, and the maximum residual limit of diazinon in vegetable products in European Union and U.S. respectively is 0.01-0.2 mg/kg and 0.05-0.7 mg/kg.

The existing common method for detecting diazinon comprises the following steps: fourier transform raman spectroscopy, spectrophotometry, chromatography, biosensors, electrochemical methods, and other quantitative detection methods. The methods have the defects of complicated sample pretreatment and determination process, high cost, requirement of professional operation and unsuitability for screening a large number of samples. The enzyme-linked immunosorbent assay based on the specific reaction of the antigen and the antibody has the advantages of high sensitivity, strong specificity, low requirements on instruments and equipment, relatively simple pretreatment of samples and the like, and is suitable for market monitoring and field monitoring. 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 diazinon has important significance for realizing nondestructive, rapid and online detection of pesticide diazinon residue.

The nano antibody is a novel genetic engineering antibody. Heavy chain antibodies that naturally lack a light chain were found in Bactria in 1993 by Belgian scientists in Bactriaceae. Later, people express the antigen binding region of the heavy chain antibody through genetic engineering technology, and the heavy chain antibody fragment with a single structural domain is the minimum antibody fragment with antigen recognition and binding capacity, which is prepared by people through genetic engineering means at present, has a molecular weight of only about 15kD, which is only about one tenth of that of the traditional antibody, and has a size at the nanometer level, so the heavy chain antibody fragment is also called as a "nano antibody" (VHH). The novel antibody has the characteristics of low immunogenicity, high stability, high affinity, high specificity and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a diazinon nano antibody and application thereof. The nano antibody can be prepared in a large scale by a gene engineering recombination expression mode. The gene engineering recombination expression mode is to clone the gene encoding 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 a protein form.

The first purpose of the invention is to provide a nano antibody for specifically recognizing diazinon.

The second purpose of the invention is to provide a gene for coding the nano antibody which can specifically recognize diazinon. .

The third object of the present invention is to provide a recombinant vector.

It is a fourth object of the present invention to provide a recombinant cell.

The fifth purpose of the invention is to provide the application of one or more of the nano antibody, the coding gene, the recombinant vector and/or the recombinant cell in the diazinon immunological detection and/or the preparation of an immunological detection kit for diazinon.

It is a sixth object of the invention to provide a method for detecting diazinon for non-diagnostic purposes.

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

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

the invention constructs a bactrian camel immune antibody library, coats a detection antigen solid phase on an enzyme label plate by using a phage display technology, and puts the bactrian camel immune antibody library into the enzyme label plate for affinity panning to obtain a nano antibody specifically combined with the diazinon resistance, wherein the nano antibody has an amino acid sequence shown in SEQ ID NO. 1. And the method is applied to immunoassay of diazinon, and a rapid, sensitive and stable detection method of diazinon is established through the immunoassay.

Accordingly, the invention claims the following:

a nano antibody for specifically recognizing diazinon has an amino acid sequence shown in SEQ ID NO. 1.

The framework region (FR1-FR4) of the amino acid sequence of the nano antibody is sequentially shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ 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.

In particular, the amount of the solvent to be used,

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

EVQLLESGGGSVQAGSSLRLSCAGSGSNAPGVCVRWFRQTPGNDREWVATIDSTG YTAYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYCAARISWGLRVSCDGDFP YWGEGTQVTVSS；

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

EVQLLESGGGSVQAGSSLRLSCAGS；

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

VRWFRQTPGNDREWVAT；

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

AYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYC；

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

WGEGTQVTVSS；

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

GSNAPGVC；

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

IDSTGYT；

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

AARISWGLRVSCDGDFPY。

the gene of the nano antibody for coding specificity recognition diazinon has the amino acid sequence shown in SEQ ID No. 9.

A recombinant vector to which the coding gene is linked.

Preferably, the recombinant vector is an expression vector.

More preferably, the expression vector is pComb3 xss.

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

Preferably, the recombinant cell is an escherichia coli cell.

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

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

Preferably, the detection is carried out based on an indirect ELISA method, the diazinon complete antigen obtained by coupling the diazinon hapten shown in the formula (II) with carrier protein 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 Ovalbumin (OVA).

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

more preferably, a sample to be detected and a nano antibody with an amino acid sequence shown as SEQ ID NO.1 are added into the solid phase carrier coated with the detection antigen, and after full reaction, 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 example, TMB developing solution mixed with developing solution A and B undergoes developing reaction with 10% H₂SO₄(v/v) the reaction was terminated.

And a kit for detecting diazinon, which contains the nano antibody.

Preferably, the kit is an indirect competitive ELISA kit, the diazinon hapten and carrier protein of the formula (II) are coupled to obtain the complete antigen of the diazinon, the complete antigen of the diazinon is coated on a solid phase carrier to be used as a detection antigen, the nano antibody is used as a detection antibody for detection,

more preferably, the solid phase carrier is an enzyme label plate.

More 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 solution A and color-developing solution B.

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

Even 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 invention discloses a nano antibody which can be applied to the actual sample detection of diazinon residues. The nano antibody has the advantages of strong specificity, high temperature resistance, acid and alkali resistance, easiness in storage, low production cost, short production period and the like, and can be used as a novel material for diazinon immunodetection. 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 shows the trend of the change of serum titer and inhibition rate in the process of diazinon immunization

FIG. 2 is an SDS-PAGE picture of the diazinon nanobody NbEQ 1.

Fig. 3 is an indirect competition ELISA standard curve established based on the nanobody NbEQ 1.

Fig. 4 is a schematic diagram of the activity of the nanobody NbEQ1 in different concentrations of methanol, acetonitrile, and acetone.

FIG. 5 is a diagram showing the activity of the Nanobody NbEQ1 after incubation at 85 ℃ for various times.

Fig. 6 is a graph showing the activity of nanobody NbEQ1 in PBS at different pH.

Fig. 7 is a schematic diagram of the activity of nanobody NbEQ1 stored for different periods of time under different temperature conditions.

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 Artificial antigen of diazinon

The design and synthesis of diazinon haptens was accomplished by pre-laboratory work (Wu H L, Wang B Z, Wang Y, et al. monoclonal antibody-based ELISA for screening of diazinon in viral samples [ J ]. Analytical Methods,2021.), using a combination of two structural haptens: hapten A shown in structural formula (I) and hapten B shown in structural formula (II)

1. Preparation of diazine agricultural artificial antigen A-LF

Hapten A shown in structural formula (I) and Lactoferrin (LF) are coupled to form immune antigen through an active ester method, and structural formula (III) of the immune antigen

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 3.895mg of diazinon hapten A into the mixture solution, and stirring at room temperature in the dark or shaking for reaction 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. 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. The coupled diazinon A-LF is used as an immune antigen.

2. Preparation of diazinon artificial antigen B-BSA

Hapten B shown in structural formula (II) and Bovine Serum Albumin (BSA) are coupled by a covalent coupling method to form an artificial detection antigen shown in structural formula (IV)

The specific operation method comprises the following steps: 6.698mg of diazinon hapten B are weighed out and 0.6mL of DMF is added to dissolve the solid. 50mg of BSA was weighed and added to 5mL of carbonate buffer (pH 9.4) to prepare a 10mg/mL protein solution. The hapten B solution was added to the BSA solution and stirred overnight at room temperature. 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. And the coupled diazinon B-BSA serves as a detection antigen.

Example 2 construction of Bactrian camel immune antibody library

First, experiment method

1. Bactrian camel immunization protocol

Animal immunization is carried out on healthy bactrian camel, the complete antigen diazinon A-LF with the structural formula shown in (III) 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 of the immunizing antigen 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.

From the second immunization, 10mL bactrian camel blood is taken one week after each immunization to separate serum for detecting the immune response condition. One week after the third, fourth and fifth immunizations, 50mL bactrian camel peripheral blood was taken to separate lymphocytes for later use.

2. Monitoring of immune response status

The immune response condition is monitored by adopting an indirect competitive ELISA method, which comprises the following specific operations:

detection of antigen immobilization: the complete antigen diazinon B-BSA shown in structural formula (IV) is used as a detection antigen, and a coating solution (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Water is added to the solution to reach a constant volume of 250mL), the solution is diluted to 1 mu g/mL, added into a 96-well enzyme label plate, each well is 100 mu L, and the solution is kept stand at 4 ℃ overnight. The next day the plate was washed twice with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20) and blotted dry on absorbent paper. mu.L of 1% BSA-PBS (w/v) was added to each well for blocking, and the mixture was allowed to stand at 37 ℃ for 2 hours. Pouring out liquid in the holes, and inverting in a drying oven at 37 ℃ for 30-60 min. The dried ELISA plate can be directly used for ELISA detection, and can also be put into a sealed bag and stored at 4 ℃.

② immune reaction: dioxazinone standard stock solution was diluted to 1. mu.g/mL with PBS, and the bactrian camel serum was diluted 1000-fold first, then diluted two-fold with a total of 7 concentration gradients. Taking two rows (8-hole) of enzyme label plate micropores, and adding 50 mu L PBS into each hole of one row to be used as a titer row; one column was added 50 μ L of diluted diazinon standard per well as the "inhibition column". The diluted serum with a series of concentrations is respectively added into a titer column and an inhibition column according to the concentration order, each concentration is respectively added into a titer hole and an inhibition hole, each hole is 50 mu L, and finally, the total volume of the two holes is supplemented to 100 mu L by PBS to be used as a blank control group. After the above systems were mixed, incubated at 37 ℃ for 30min, washed 5 times with 20-fold diluted wash solution PBST (0.01M PBS, 0.05% Tween-20), and blotted dry on absorbent paper.

Adding enzyme-labeled secondary antibody: Anti-VHH-HRP (GenScript A01861-200) secondary antibody was diluted 5000-fold with PBST, 100. mu.L per well, incubated at 37 ℃ for 30min, washed 5 times with 20-fold diluted wash PBST (0.01M PBS, 0.05% Tween-20), and blotted dry on absorbent paper.

And fourthly, TMB substrate color reaction and termination: TMB substrate solution A and B (Solarbio, PR1210) were mixed in equal volumes in advance, 100. mu.L was added to each well, incubation was carried out at 37 ℃ for 10min, and 50. mu.L was added to each well to terminate the reaction.

Reading and data analysis: reading an absorbance value (OD450nm) under 450nm by using a microplate reader, defining the corresponding serum dilution multiple when the OD value of the titer column is between 1 and 1.5 as a serum titer (if more than one reading is in the interval, a value close to 1 is taken), and defining the reduction degree of the reading of the inhibition hole corresponding to the titer hole as an inhibition rate, and calculating by using the following formula. And selecting the lymphocyte with the highest titer and inhibition rate to prepare the nano antibody according to the immune response result.

3. Isolation of Bactrian camel lymphocytes

Mixing the whole blood of the bactrian camel with isometric physiological saline in a volume ratio of 1:1 to obtain diluted blood, and standing at normal temperature. 20mL of lymph isolate was added to a sterile 50mL centrifuge tube and 20mL of diluted blood was added slowly along the tube wall using a sterile pasteur dropper. Centrifuge at 500g for 30 min. The lymphocyte layer was taken out to a new 50mL centrifuge tube, diluted 2-fold with physiological saline, centrifuged at 2000g at 4 ℃ for 10min, and the supernatant was discarded. The lymphocytes were blown off with 5mL of physiological saline, centrifuged again at 2000g for 10min, and the supernatant was discarded to sufficiently wash the lymphocytes. Lysis solution (TRNsol) was added to each lymphocyte, and 1mL aliquots were dispensed into 2mL centrifuge tubes and stored at-80 ℃ until use.

4. Extraction of Total RNA

The total RNA extraction was performed according to the Trizol reagent method of Invitrogen corporation. The specific method comprises the following steps:

0.2mL of chloroform was added to 1mL of the above lysate. The centrifuge tube lid was closed, shaken vigorously for 15 seconds, and incubated on ice for 5 min. 12000 at room temperatureCentrifuge at rpm for 10 min. Transferring not more than 80% of the upper aqueous phase to a new centrifuge tube, slowly adding 0.7 volume times of anhydrous ethanol, and mixing; transferring the obtained solution and the precipitate into a GBC adsorption column, centrifuging at 12000rpm for 30 s, and discarding the waste liquid; adding 500 mu L of Wash Buffer I into the GBC adsorption column, centrifuging for 1min, and discarding the waste liquid; add 600. mu.L of Wash Buffer II to GBC adsorption column at 12000rpm, centrifuge for 30 seconds, and discard waste. Centrifuging at 12000rpm for 1min, discarding waste liquid, and opening the cover at room temperature to air-dry the residual rinsing liquid in the adsorption column. Transferring the GBC adsorption column into a new centrifuge tube, and adding 30-100 mu L dd H₂O, standing at room temperature for 2min, and centrifuging at 12000rpm at 4 ℃ for 1 min. The liquid in the tube was collected and stored at-80 ℃.

5. Synthesis of cDNA

First strand cDNA synthesis was performed using RNA as a template, according to the Takara first Strand reverse transcription kit instructions. The specific method comprises the following steps:

(1) according to the first reaction system of cDNA synthesis shown in Table 1, the reagents were mixed in a nuclease-free centrifuge tube and operated in an ice bath;

TABLE 1 first step reaction System for cDNA Synthesis

Total RNA	3μg
		Oligo(dT)₁₈primer	1μL
RNase free ddH₂O	Up to12μL
		Total	12μL

(2) Incubating the reaction system at 65 ℃ for 5min, and cooling for 2min in an ice bath;

(3) according to the second reaction system of cDNA synthesis shown in Table 2, adding reagent into the system after the previous reaction;

TABLE 2 second step reaction System for cDNA Synthesis

The system after the last step of reaction	12μL
			5×Reaction Buffer	4μL
RiboLock RNase Inhibitor(20U/μL)	1μL
		10mM dNTP Mix	2μL
RevertAid M-MiLVRT(200U/μL)	1μL
		Total	20μL

(4) Incubation was carried out at 42 ℃ for 60min and at 70 ℃ for 5 min. The reverse transcription product cDNA was stored at-80 ℃.

6. 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 3 primer sequences for the Bactrian camel heavy chain antibody genes

The first round of PCR uses cDNA as its PCR template, and the specific reaction parameters are shown in tables 4 and 5:

TABLE 4 nested PCR first-step reaction System

TABLE 5 nested PCR first step reaction conditions

94℃	5min
		94℃	30s
55℃	30s
		72℃	1min,go to step2,30cycle
72℃	10min
		4℃	Forever

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 carried out using the recovered product of the first round PCR as a template, and the specific reaction parameters are shown in tables 6 and 7

TABLE 6 nested PCR second-step reaction System

TABLE 7 nested PCR second step reaction conditions

7. Gene library construction

(1) Digestion of VHH target genes and vectors

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; and directly and cleanly recovering the enzyme digestion product of the VHH gene by 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 90mm as a counting plate, and the bacteria liquid is cultured overnight at 37 ℃. And (3) coating all the rest undiluted resuscitation bacterial liquid on LB-Amp culture dishes with the diameter of 120mm, coating 2-3 culture dishes per 1mL bacterial liquid as amplification plates, and performing amplification culture at 37 ℃ overnight.

Counting the number of bacterial colonies on a counting culture dish, calculating the total number of bacteria in the resuscitation bacteria liquid, and carrying out multiple electric shock transformation to ensure that the total number of transformed bacterial colonies is accumulated to 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 25% glycerol (v/v) to the final concentration, performing gradient dilution on 50 mu L of bacterial liquid to determine the cell number, subpackaging the rest of bacterial liquid, and freezing and storing at-80 ℃ to obtain the diazinon nano antibody gene library.

8. Phage rescue

Inoculating cells with more than 10 times of library volume into 200mL LB (Amp) at 37 ℃, culturing at 250rpm until the OD600 is about 0.4-0.6; the helper phage M13K07(20:1 multiplicity of infection) was added, left to stand at 37 ℃ for 30min, and then cultured at 250rpm for 1h, and kanamycin antibiotic (1: 1000) was added and cultured at 37 ℃ and 250rpm overnight. Centrifuging at 12000rpm at 4 ℃ for 15min, taking the supernatant, adding 1/5 volumes of PEG/NaCl (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. Centrifuging at 12000rpm for 15min at 4 deg.C, discarding supernatant, resuspending the precipitate with 1mL TBS, transferring to 2mL centrifuge tube, centrifuging at 12000rpm for 5min at 4 deg.C, filtering with 0.22 μm polyethersulfone filter membrane, collecting 10 μ L of assay reservoir, adding glycerol with final concentration of 50%, and preserving at-80 deg.C.

Second, experimental results

The immunogen is used for bactrian camel immunity, and the detection antigen is used for monitoring immune response. The bactrian camel immunity is performed for 5 times in total, the immune response situation is shown in figure 1, from three-time immunity, the bactrian camel serum titer is remarkably improved compared with the negative serum before immunity, the titer reaches 1:2000, and the inhibition rate is close to 80%; the immune response is stable in the four-immunity and five-immunity, the titer is 1:4000, and the inhibition rate is about 89%. Therefore, blood from the fourth and fifth immunizations is collected for pooling.

Example 3 affinity panning and identification of Nanobodies

First, experiment method

1. Affinity panning of Nanobodies

Coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the oxazine artificial antigen B-BSA shown in the structural formula (IV) prepared in example 1 to 1 mu g/mL, adding the diluted oxazine artificial antigen B-BSA into micropores of an ELISA plate, keeping the diluted oxazine artificial antigen B-BSA at a temperature of 100 mu L per hole, and standing the diluted oxazine artificial antigen B-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.

BSA was added to the above diazinon nanobody phage library to make the final concentration of BSA 1% (w/v), and the phage library (w/v) containing 1% BSA was added to 3 wells containing immobilized antigen, 100. mu.L of each well was added, and incubated at 37 ℃ for 1 h. Unbound phage in wells were discarded, wells were washed 10 times with PBST and 5 times with PBS. Adding a diazinon standard substance with the concentration of 5 mu g/mL into each hole, adding 100 mu L of the diazinon standard substance into each hole, performing shake incubation for 4 hours at 37 ℃ to perform competitive reaction, and collecting the liquid in the micropores into a sterile centrifuge tube. The phage at this point are called "competing outputs" and the first round of screening is complete. mu.L of eluted phage was titered and the remainder was used to infect 4mL of E.coil TG1 strain grown to log phase for amplification. On the third day, amplified phages were precipitated with 5-fold diluted PEG/NaCl solution (100g of PEG 8000 and 73.05g of sodium chloride, made up to 500mL with water) and the titer of the phages was determined.

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 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

Bag for childrenQuilt liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Water was added to a constant volume of 250mL) the diazinon artificial antigen B-BSA represented by the structural formula (IV) prepared in example 1 was diluted to 1. mu.g/mL and allowed to stand overnight at 4 ℃. 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 per well, sealing at 37 deg.C for 2 hr, discarding sealing solution, oven drying at 37 deg.C for 60min, and storing at 4 deg.C for use.

(2) Nanobody miniexpression

Third, four rounds of panning competitive elution output titer assay plate randomly selected 30 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

And (3) centrifuging the deep-well plate at 4000rpm for 20min, taking two enzyme-labeled plates with immobilized antigens, adding 50 mu LPBS into each hole of the plate 1, adding PBS containing 10ng/mL diazinon standard drugs into the plate 2, sucking supernatant from the centrifuged 96-well plate, and adding 50 mu L into each hole of the enzyme-labeled plate with the corresponding number. Incubate at 37 ℃ for 30min, wash five times with 20-fold diluted wash PBST (0.01MPBS, 0.05% Tween-20), and pat dry the well contents. Anti-VHH-HRP (GenScript, A01861-200) secondary antibody 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, patting off the liquid in the well, adding 100 μ L of TMB developing solution mixed with equal volume of developing solution A and developing solution B (Solarbio, PR1210) into each well, and developing at 37 deg.C for 10 min; adding 50 mu L of stop solution 10% H₂SO₄(v/v) terminating the 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

The phage clone of the nano antibody obtained by indirect competition ELISA identification is sent to a sequencing company for gene sequencing, and the amino acid sequence of the nano antibody can be obtained according to the DNA sequencing result and a codon table. The result shows that 1 strain diazinon nano antibody is obtained and is named as NbEQ 1.

NbEQ1 has an amino acid sequence shown in SEQ ID NO.1, and the nanobody comprises 4 FR framework regions and 3 CDR complementarity determining regions which are arranged in the sequence of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, wherein the framework regions (FR1-FR4) are sequentially shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ 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.

The amino acid sequence of VHH of the nano antibody NbEQ1 is shown in SEQ ID NO. 1:

the amino acid of the framework region FR1 of the nanometer antibody NbEQ1 is shown in SEQ ID NO. 2:

EVQLLESGGGSVQAGSSLRLSCAGS；

the amino acid of the framework region FR2 of the nano antibody NbEQ1 is shown in SEQ ID NO. 3:

VRWFRQTPGNDREWVAT；

the amino acid of the framework region FR3 of the nano antibody NbEQ1 is shown in SEQ ID NO. 4:

AYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYC；

WGEGTQVTVSS；

the amino acid of the CDR1 of the NbEQ1 is shown in SEQ ID NO. 6:

GSNAPGVC；

the amino acid of the CDR2 of the NbEQ1 is shown in SEQ ID NO. 7:

IDSTGYT；

the amino acid of the CDR3 of the NbEQ1 is shown in SEQ ID NO. 8:

AARISWGLRVSCDGDFPY。

meanwhile, the nucleotide sequence of the gene for coding the NbEQ1 is shown as SEQ ID No. 9.

EXAMPLE 4 Mass production of Nanobody NbEQ1

The method for preparing the nano antibody NbEQ1 in a protein expression mode comprises the following steps:

the obtained phage clone of the nano antibody NbEQ1 is extracted by a kit, and the plasmid is transferred into E.coil BL21 by a chemical transformation method. A single colony from the transformation plate was inoculated in 10mL of LB (Amp) medium and cultured at 37 ℃ and 250rpm overnight. The overnight cultures were incubated at 1: 100 (v/v) was inoculated into 750mL of LB (Amp) medium and cultured at 37 ℃ and 250rpm until the OD600 was about 0.4 to 0.6, and IPTG was added to a working concentration of 1mM and cultured at 37 ℃ and 250rpm overnight. Centrifuging at 12000rpm for 5min at 4 deg.C in the next day, collecting thallus precipitate, centrifuging at 12000rpm for 10min by sucrose osmotic freeze thawing method, collecting supernatant, and purifying the supernatant by affinity chromatography to obtain nanometer antibody NbEQ1 (shown in SEQ ID NO. 1) shown in FIG. 2.

Example 5 determination of working concentration and sensitivity of Nanobody NbEQ1

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), diluting the diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mu g/mL, adding the diluted antigen B-BSA into micropores of an ELISA plate, keeping the diluted antigen in each hole by 100 mu L, and standing the antigen at 4 ℃ overnight. Diluting the solution the next dayAfter washing the plate twice with 20-fold 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 and the solution stored at 4 ℃ after drying on absorbent paper and drying at 37 ℃ for 1 h.

2. Determination of working concentration of antibody

The purified nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 is diluted by PBS for two-fold gradient dilution to obtain a series of nano antibodies with different concentrations. mu.L PBS per well was added to the microplate, and then 50. mu.L of the antibody diluted in a gradient was added thereto, and 3-well replicates were performed for each antibody concentration, while a blank (100. mu.L PBS) was performed in 3 wells. Incubation was carried out at 37 ℃ for 30min, plates 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 (GenScript, A01861-200) secondary antibody diluted 5000-fold with PBST was added to each well after blotting onto absorbent paper. Incubating at 37 deg.C for 30min, washing the plate 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 into each well, incubating at 37 deg.C for 10min in 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 above experimental conditions, the working concentration of NbEQ1 is 91.5 ng/mL.

3. Indirect competitive ELISA standard curve drawing

Diazinon standards were diluted with PBS to give a series of diazinon solutions of different concentrations, added to the elisa plate at 50 μ L per well, 3-well replicates were performed at each concentration, and a 3-well drug blank (50 μ L PBS) was prepared. The nanobody NbEQ1 having the amino acid sequence shown in SEQ ID No.1 was diluted with PBS to a working concentration of 91.5ng/mL, and 50. mu.L of the diluted antibody was added to each well. Incubation was carried out at 37 ℃ for 30min, plates 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 (GenScript, A01861-200) secondary antibody diluted 5000-fold with PBST was added to each well after blotting onto absorbent paper. Incubating at 37 deg.C for 30min, washing the plate with 20-fold diluted washing solution PBST (0.01M PBS, 0.05% Tween-20) for 5 times, drying on absorbent paper, and adding 100 μ L of TMB color developing solution into each wellIncubating at 37 deg.C in dark for 10min, adding 50 μ L stop solution 10% H per well₂SO₄(v/v), reading OD450nm on a microplate reader.

The average value of OD450 values of the drug blank group is recorded as B₀The average OD450 values at different drug concentrations are denoted as B_xCalculating B by Excel under different drug concentrations_x/B₀Ratios and standard deviations for each set of parallel data. With drug concentration as abscissa, B_x/B₀And 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.

Second, experimental results

An indirect competition ELISA standard curve graph established based on an antibody NbEQ1 with an amino acid sequence shown as SEQ ID NO.1 is shown in FIG. 3, and the standard curve is S-shaped, the linear correlation is good, the detection range is 2.154 ng/mL-12.031 ng/mL, the IC50 is 5.091ng/mL, the minimum limit of detection (LOD) is 0.161ng/mL, and the detection requirement that the maximum residual limit of China is 0.01mg/kg can be met.

Example 6 detection method of diazinon

1. Pre-coating a plate: coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in example 1 to 1 mu g/mL, adding the diluted antigen B-BSA into micropores of an ELISA plate, keeping the diluted antigen in each hole by 100 mu L, and standing the antigen 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, allowed to stand at 37 ℃ for 2 hours, the liquid in the wells was poured out, blotted on absorbent paper and baked at 37 ℃ for 1 hour, and then stored at 4 ℃ for 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 nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 is diluted to the working concentration of 91.5ng/mL by PBS, 50 mu L of diluted antibody solution is added into all the holes, and the mixture is incubated at 37 DEG CAnd culturing 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 patted 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 PBST (0.01M PBS, 0.05% Tween-20) diluted 20-fold and blotted dry on absorbent paper.

5. mu.L of TMB developing solution prepared by mixing equal volumes of developing solution A and developing solution B (Solarbio, PR1210) 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.

Third, interpretation of results

The average value of blank OD450nm values of the standard is recorded as B₀The average value of OD450nm and the wells to be tested of the samples at different drug concentrations is recorded as B_xThe Bx/B0 ratios for different drug concentrations or sample wells were calculated as well as the 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 can be obtained, namely the logarithm value of the concentration of diazinon, and the antilog is obtained, namely the concentration of diazinon 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 7 detection kit for diazinon

Composition of kit

1. Pre-coating a plate: coating with coating liquid (0.375g Na)₂CO₃With 0.7325g of NaHCO₃Adding water to a constant volume of 250mL), diluting the diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in example 1 to 1 mu g/mL, adding the diluted antigen B-BSA into micropores of an ELISA plate, keeping the diluted antigen in each hole by 100 mu L, and standing the antigen 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. Pouring out the liquid in the hole, patting dry on absorbent paper and 3Baking at 7 deg.C 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: diazinon standard solutions of different concentrations.

4. Dilutions of antibody and sample: PBS.

5. Dilution of labeled antibody: PBST.

6. Developing solution A and solution B.

7. Stopping liquid: 10% H₂SO₄(v/v)。

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

9. Detecting an antibody: the amino acid sequence of the nano antibody NbEQ1 is 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 NbEQ1 having the amino acid sequence shown in SEQ ID No.1 was diluted to the working concentration of 91.5ng/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 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 for 5 times by using washing liquor PBST (0.01M PBS, 0.05% Tween-20) diluted by 20 times, beating the micropores dry on absorbent paper, adding 100 mu L of TMB developing solution formed by mixing equal-volume developing solution A and developing solution B into each hole, incubating for 10min in a dark place 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 OD450nm and the wells to be tested of the samples 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 diazinon, and the antilog is obtained, namely the concentration of diazinon 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 the Nanobody NbEQ1

First, experiment method

Other 12 analogs of diazinon (pirimiphos-methyl, Diazoxon, isocarbophos, quinalphos, fenitrothion, methyl parathion, ethyl parathion, methidathion, triazophos, phoxim, profenofos) and the diazinon metabolite (IMHP) were tested using the kit of example 7.

Standard solutions of various diazinon analogs were prepared, and a standard curve was drawn for each diazinon analog and metabolite according to the various diazinon analogs tested at a gradient concentration using the kit of example 7 to give respective IC50 values. The cross-reactivity of each drug with nanobody NbEQ1 was calculated using the following formula:

second, experimental results

The results are shown in table 8, the cross-reactivity rates of the nano antibody NbEQ1 with diazinon analogs such as pirimiphos-methyl and Diazoxon, the cross-reactivity rates of which are respectively 6.877% and 1.324% and the cross-reactivity rates of which are lower than 1% with other analogs (IC50 is greater than 500ng/mL), which indicates that the nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 can specifically recognize diazinon, and the established detection method and detection kit have higher specificity for diazinon detection.

TABLE 8 sensitivity and specificity of diazinon Nanobody NbEQ1

Example 9 Activity of Nanobody NbEQ1 in different concentrations of organic solvent

First, experiment method

The nanobody NbEQ1, the amino acid sequence of which is shown in SEQ ID No.1, was diluted with PBS to a working concentration of 91.5 ng/mL. Solutions of methanol, acetonitrile and acetone were prepared in different volume ratios (0%, 20%, 40%, 60%, 80%, 100%) with PBS. Each well was first filled with 50. mu.L of organic solvent solutions of different volume ratios, and then 50. mu.L of diluted antibody solution was added. The detection kit in example 7 is used to respectively determine the binding capacity of the antibody and the detection antigen under different organic solvent conditions, and the tolerance capacity of the nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID No.1 to different organic solvents is 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 data were normalized by taking the titer at 0 in the organic solvent as 100%. The determination result is shown in fig. 4, the binding activity of the nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 is reduced along with the increase of the concentration of the organic solvent, and particularly the reduction trend is fastest in the organic solvent of acetonitrile. At an acetonitrile concentration of 10%, the nanobody NbEQ1 has lost 60% of its antigen binding activity. When the proportion of methanol, acetonitrile and acetone in the organic solvent mixed solution reaches 30%, 20% and 30% respectively, the antibody is completely inactivated. The nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID No.1 has poor tolerance to three organic solvents commonly used in pretreatment of methanol, acetonitrile and acetone in a standard method, particularly cannot tolerate acetonitrile, and the organic solvent should be removed in an antibody working solution as much as possible to avoid the influence of a high-concentration organic solvent on the sensitivity of the antibody.

Example 10 Activity of Nanobody NbEQ1 at different temperatures

First, experiment method

The nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 was diluted to the working concentration (91.5ng/mL) with PBS, and incubated at 85 ℃ for different times (0, 10, 20, 30, 40, 50, 60 min). Using the kit of example 7, the binding activity of the antibody to the test antigen after the high-temperature treatment for various times was measured, respectively.

Second, experimental results

The data were normalized by using the titer with a heating time of 0 as 100%. The determination result is shown in fig. 5, and the binding activity of the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 to the antigen binding activity to be detected is in a decreasing trend with the increase of the incubation time. The nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 still has more than 35% of antigen binding activity even after being heated for 1 hour at 85 ℃. The characteristics show that the nano antibody NbEQ1 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 characteristics can enable the nano antibody NbEQ1 to have greater application advantages in a higher-temperature detection environment.

Example 11 Activity of Nanobody NbEQ1 at different pH conditions

First, experiment method

The solvent of the nanobody NbEQ1, the amino acid sequence of which is shown in SEQ ID No.1, was diluted to working concentration (91.5ng/mL) with 0.01M PBS as diluent at different pH values (2.4, 3.4, 5.4, 6.4, 7.4, 8.4, 9.4, 10.4), respectively. The kit of example 7 was used to determine the binding ability of the antibody to the test antigen and to evaluate the binding activity of the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 under different pH conditions.

Second, experimental results

Data were normalized to titer at pH 7.4 as 100%. The determination result is shown in fig. 6, in the range of ph 3.4-10.4, the antigen binding activity of the nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 is gradually reduced, and it is noted that under acidic conditions, the antigen binding activity is higher. When the pH drops to 2.4, the antibody is substantially inactivated. In general trend, the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 can maintain good antigen binding activity in a wide pH range, but is more suitable for working in an acidic environment with a pH of not less than 3.4.

Example 12 storage stability of Nanobody NbEQ1 at different temperature conditions

First, experiment method

Adding 0.03% preservative ProClin 300(v/v) and 0.01% protease inhibitor (v/v) into a nano antibody NbEQ1 solution with an amino acid sequence shown as SEQ ID NO.1, subpackaging 20 mu L of each part, and respectively placing the parts in four temperatures (-20, 4, 25 and 37 ℃) which are most common in daily use to be protected from light for storage. The kit of example 7 was used to determine the binding ability of the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 to the test antigen after storage for different time periods (0, 7, 14, 30, 60Day) at different temperatures.

Second, experimental results

The storage stability of the antibody added with the preservative and the protease inhibitor is detected so as to determine the shelf life of the nano antibody with the amino acid sequence shown as SEQ ID NO. 1. The data were normalized by taking the titer at 0day of storage as 100%. As shown in FIG. 7, the antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 still has 62%, 50% and 60% of antigen binding activity after being stored at-20 ℃ and 4 ℃ for two months at normal temperature, even if it is stored at higher temperature (37 ℃) for about half a month, the antibody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 still has 37% of antigen binding activity, and it is noted that the titer does not decrease but increases when the storage time reaches more than one month at 37 ℃, which may be caused by the concentration of the antibody solution becoming high due to evaporation. In summary, the nanobody NbEQ1 has a certain storage stability, can be transported, stored and used for a long time at room temperature after adding a preservative or protease inhibitor, and can be stored for a short time even at a relatively high temperature of 37 ℃.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. 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> nano antibody for detecting diazinon and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Ser Asn Ala Pro Gly Val

20 25 30

Cys Val Arg Trp Phe Arg Gln Thr Pro Gly Asn Asp Arg Glu Trp Val

35 40 45

Ala Thr Ile Asp Ser Thr Gly Tyr Thr Ala Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Lys Glu Asp Ala Lys Arg Thr Val Tyr Leu

65 70 75 80

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

85 90 95

Ala Arg Ile Ser Trp Gly Leu Arg Val Ser Cys Asp Gly Asp Phe Pro

100 105 110

Tyr Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser

20 25

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Arg Trp Phe Arg Gln Thr Pro Gly Asn Asp Arg Glu Trp Val Ala

1 5 10 15

Thr

<210> 4

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Lys Glu Asp

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 5

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Ser Asn Ala Pro Gly Val Cys

1 5

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ile Asp Ser Thr Gly Tyr Thr

1 5

<210> 8

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ala Ala Arg Ile Ser Trp Gly Leu Arg Val Ser Cys Asp Gly Asp Phe

1 5 10 15

Pro Tyr

<210> 9

<211> 372

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaggtgcagc tgctggagtc tgggggaggc tcggtgcagg ctggaagttc tctgagactc 60

tcctgtgcag ggtctggttc gaacgccccg ggtgtctgcg tgcgctggtt ccgtcagact 120

ccagggaacg accgcgagtg ggtcgcgact attgactcta ctggatacac ggcctacgca 180

gactccgtga agggccggtt cactgtctcc aaagaggacg ccaagcgcac tgtgtatctg 240

caaatgaaca ggctgagacc tgaggacact gccatgtact attgtgcggc aaggatatct 300

tggggccttc gggtatcgtg cgatggtgac tttccttact ggggagaggg gacccaggtc 360

accgtctcct ca 372

Claims

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

2. A gene of a nano antibody for coding specificity recognition of diazinon is characterized in that the amino acid sequence of the gene is shown as SEQ ID NO. 9.

3. A recombinant vector comprising the coding 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 coding gene of claim 2, the recombinant vector of claim 3, and/or the recombinant cell of claim 4 in the detection of diazinon and/or in the preparation of an immunological detection kit for diazinon.

6. A method for detecting diazinon for non-diagnostic purposes, comprising using the nanobody of claim 1.

7. The detection method according to claim 6, wherein the detection is carried out based on an indirect ELISA method, wherein a diazinon complete antigen obtained by coupling a diazinon hapten represented by formula (II) with a carrier protein is used as a detection antigen, the nano-antibody is used as a detection antibody,

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

9. The kit of claim 8, wherein the nanobody is coated on a solid phase carrier, and further comprises a diazinon complete antigen obtained by coupling the diazinon hapten and carrier protein in the formula (II) as a detection antigen,

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