CN114409795B - Nanometer antibody for detecting diazinon and application thereof - Google Patents

Nanometer antibody for detecting diazinon and application thereof Download PDF

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CN114409795B
CN114409795B CN202111604801.5A CN202111604801A CN114409795B CN 114409795 B CN114409795 B CN 114409795B CN 202111604801 A CN202111604801 A CN 202111604801A CN 114409795 B CN114409795 B CN 114409795B
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徐振林
黄丹彤
何晓婷
陈子键
王弘
沈玉栋
雷红涛
罗林
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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 for encoding the nano antibody is shown as SEQ ID NO. 9. The invention discloses a nano antibody which can be applied to actual sample detection of diazinon residues. The nano antibody has the advantages of strong specificity, high temperature resistance, acid and alkali resistance, easy preservation, low production cost, short production period and the like, can be used as a novel material for detecting diazinon, and has good application prospect and wide development space in the rapid and effective detection of the diazinon.

Description

Nanometer antibody for detecting diazinon and application thereof
Technical Field
The invention relates to the technical field of pesticide detection, in particular to a diazinon nanobody and application thereof.
Background
Diazinon (DAZ) is a broad-spectrum and high-efficiency organophosphorus insecticide, has the functions of contact killing, stomach toxicity, fumigation and certain systemic action, and is used for preventing and controlling ectoparasitic diseases of livestock mites, flies, lice, ticks and other important crops such as rice, cotton, vegetables, flowers and the like. However, due to the great use of diazinon and the limitations of application technology, diazinon can remain widely in environmental media such as water, soil and food, and enter various organisms and human bodies through the food chain. 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 range of syndromes including anorexia, diarrhea, general weakness, muscle tremors, postural and behavioral abnormalities, depression, death, and the like. Therefore, a stricter maximum residual limit is set for diazinon residues in foods worldwide, for example, the maximum residual limit of diazinon in foods in China is 0.01-0.5 mg/kg, and the European Union and the United states respectively prescribe that the maximum residual limit of diazinon in vegetable products 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 complicated sample pretreatment and measurement processes, high cost, require professional operation and are not suitable for screening a large number of samples. The ELISA detection method based on the specific reaction of the antigen and the antibody has the advantages of high sensitivity, strong specificity, low instrument and equipment requirements, relatively simple sample pretreatment and the like, and is suitable for market monitoring and field monitoring. The key to success of the immunoassay method is to prepare antibodies with high sensitivity and monoclonal antibodies and polyclonal antibodies which are 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 method of the stable antibodies with strong specificity and high sensitivity for diazinon has important significance for realizing nondestructive, rapid and online detection of pesticide diazinon residues.
The nano antibody is a novel genetic engineering antibody. The belgium scientist in 1993 found heavy chain antibodies naturally deleted for the light chain in Bactrian animals. Later people express the antigen binding region of the heavy chain antibody by genetic engineering technology, the heavy chain antibody fragment of the single structural domain is the minimum antibody fragment with antigen recognition and binding capacity, which is prepared by genetic engineering means at present, the molecular weight of the heavy chain antibody fragment is only about 15kD, and the molecular weight of the heavy chain antibody fragment is only about one tenth of that of the traditional antibody, and the size of the heavy chain antibody fragment is in the nanometer level, so the heavy chain antibody fragment is also called as "nanobody" (Variable domain of heavy chain of heavy chain 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 provide a diazinon nanobody and application thereof. The nano antibody 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.
It is a first object of the present invention to provide a nanobody that specifically recognizes diazinon.
A second object of the present invention is to provide a gene encoding a nanobody that specifically recognizes diazinon. .
A 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 object of the invention is to provide the application of one or more of the nanobody, the coding gene, the recombinant vector and/or the recombinant cell in the detection of diazinon immunology and/or the preparation of diazinon immunology detection kit.
It is a sixth object of the present invention to provide a method for detecting diazinon for non-diagnostic purposes.
The seventh object of the invention is to provide a kit for detecting diazinon.
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 for coating a detection antigen solid phase 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 an anti-diazinon specific binding nanobody is obtained, and the nanobody has an amino acid sequence shown as SEQ ID NO. 1. The method is applied to immunological detection analysis of diazinon, and a rapid, sensitive and stable diazinon detection method is established through the immunological detection analysis.
Accordingly, the present invention claims the following:
a specific diazinon-recognizing nano antibody has the amino acid sequence shown in SEQ ID NO. 1.
The framework regions (FR 1-FR 4) of the amino acid sequence of the nano antibody are sequentially shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ 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.
In particular, the method comprises the steps of,
the VHH amino acid sequence of the nanobody is shown in SEQ ID NO. 1:
EVQLLESGGGSVQAGSSLRLSCAGSGSNAPGVCVRWFRQTPGNDREWVATIDSTG YTAYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYCAARISWGLRVSCDGDFP YWGEGTQVTVSS；
the amino acid of the framework region FR1 of the nano antibody is shown as SEQ ID NO. 2:
EVQLLESGGGSVQAGSSLRLSCAGS；
the amino acid of the framework region FR2 of the nano antibody is shown as SEQ ID NO. 3:
VRWFRQTPGNDREWVAT；
the amino acid of the framework region FR3 of the nano antibody is shown as SEQ ID NO. 4:
AYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYC；
the amino acid of the framework region FR4 of the nano antibody is shown as SEQ ID NO. 5:
WGEGTQVTVSS；
the amino acid of the complementarity determining region CDR1 of the nano antibody is shown in SEQ ID NO. 6:
GSNAPGVC；
the amino acid of the complementarity determining region CDR2 of the nano antibody is shown in SEQ ID NO. 7:
IDSTGYT；
the amino acid of the complementarity determining region CDR3 of the nano antibody is shown in SEQ ID NO. 8:
AARISWGLRVSCDGDFPY。
a gene for coding the nano antibody for specifically recognizing diazinon has the amino acid sequence shown in SEQ ID NO. 9.
A recombinant vector, wherein the recombinant vector is connected with the coding gene.
Preferably, the recombinant vector is an expression vector.
More preferably, the expression vector is pComb3xss.
A recombinant cell comprising the expression vector or capable of expressing the nanobody.
Preferably, the recombinant cell is an E.coli cell.
The application of one or more of the nanobody, the gene, the recombinant vector and/or the recombinant cell in the detection of diazinon and/or the preparation of diazinon immunological detection kit also belongs to the protection scope of the invention.
The invention discloses a non-diagnostic target detection method of diazinon, which utilizes the nano antibody.
Preferably, the detection is carried out based on an indirect ELISA method, the diazinon hapten shown in the formula (II) and the diazinon complete antigen obtained by coupling carrier protein are used as detection antigens, the nano antibody is used as detection antibody for detection,
Figure BDA0003433342650000041
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),
Figure BDA0003433342650000042
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 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 color A and the color B are mixed with TMB color solution for color reaction, 10% H 2 SO 4 (v/v) terminate the reaction.
And a kit for detecting diazinon, comprising the nanobody.
Preferably, the kit is an indirect competition ELISA kit, the diazinon hapten obtained by coupling the diazinon hapten and carrier protein in the formula (II) 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,
Figure BDA0003433342650000043
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),
Figure BDA0003433342650000051
more preferably, the solid support is an enzyme-labeled version.
More preferably, the kit further comprises an enzyme-labeled secondary antibody, a color developing agent and a terminator.
Still more preferably, the color developer is color developer A and color developer B.
Still more preferably, the terminator is 10% H 2 SO 4 (v/v)。
Still 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 invention discloses a nano antibody which can be applied to actual sample detection of diazinon residues. The nano antibody has the advantages of 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 diazinon immunodetection. 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 shows the trend of the change of serum potency and inhibition rate in diazinon immunization
FIG. 2 is a SDS-PAGE of diazinon nanobody NbEQ 1.
Fig. 3 is an indirect competition ELISA standard curve established based on nanobody NbEQ 1.
Fig. 4 is a schematic diagram showing the activity of the nanobody NbEQ1 in methanol, acetonitrile, acetone at different concentrations.
Fig. 5 is a schematic diagram of the activity of the nanobody NbEQ1 after incubation at 85 ℃ for different times.
Fig. 6 is a schematic diagram of the activity of nanobody NbEQ1 in PBS at different pH.
FIG. 7 is a schematic diagram showing the activity of NbEQ1 of 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 diazinon Artificial antigen
The design and synthesis of diazinon hapten was completed by laboratory preliminary work (Wu H L, wang B Z, wang Y, et al monoclonal antibody-based icELISA for screening of diazinon in vegetable samples [ J ]. Analytical Methods, 2021.) and hapten of two structures was used together: hapten A shown in structural formula (I) and hapten B shown in structural formula (II)
Figure BDA0003433342650000061
1. Preparation of diazine agro-artificial antigen A-LF
Hapten A shown in structural formula (I) and Lactoferrin (LF) are coupled by an active ester method to form an immune antigen, and the immune antigen is shown in structural formula (III)
Figure BDA0003433342650000062
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. 3.895mg of diazinon hapten A 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 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. 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, wherein the artificial detection antigen is shown in structural formula (IV)
Figure BDA0003433342650000071
The specific operation method is as follows: 6.698mg of diazinon hapten B was weighed and 0.6mL of DMF was 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, packaging, and freezing at-20deg.C. Dialyzing with PBS for 3 times, packaging, and freezing at-20deg.C. The coupled diazinon B-BSA is used as a detection antigen.
EXAMPLE 2 construction of Bactrian camel immune antibody library
1. Experimental method
1. Bactrian camel immunization protocol
Animals were immunized with a healthy Bactrian camel and the complete antigen diazinon A-LF of formula (III) prepared in example 1 was used as the immunizing antigen and subcutaneously injected in the neck of the Bactrian camel 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.
From the second immunization, 10mL of the Bactrian camel blood was taken after one week of each immunization to isolate serum for detection of immune response. After the third, fourth and fifth immunization for one week, 50mL of the peripheral blood of the Bactrian camel is taken for separating lymphocytes for standby.
2. Monitoring of immune response conditions
The immune response is monitored by adopting an indirect competition ELISA method, and the specific operation is as follows:
(1) detection of antigen immobilization: the complete antigen diazinon B-BSA shown in the structural formula (IV) is used as a detection antigen, and a coating solution (0.375 g Na 2 CO 3 And 0.7325g NaHCO 3 Water was added to volume 250 mL) and diluted to 1 μg/mL, added to 96-well elisa plate, 100 μl per well and left to stand overnight at 4 ℃. The enzyme-labeled plate was washed twice with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) the next day, and was dried on absorbent paper. mu.L of 1% BSA-PBS (w/v) was added to each well The rows are closed and kept stand at 37 ℃ for 2 hours. Pouring out the liquid in the hole, and inverting the liquid in an oven at 37 ℃ for 30-60 min. The dried ELISA plate can be directly used for ELISA detection or can be put into a sealed bag for storage at 4 ℃.
(2) Immune response: the diazinon standard stock was diluted to 1 μg/mL with PBS, the serum of the alpaca was diluted 1000-fold and then subjected to two-fold gradient dilution for a total of 7 concentration gradients. Taking two rows (8 holes) of ELISA plate micropores, adding 50 mu L of PBS into each hole of one row, and taking the PBS as a titer row; one column of diluted diazinon standard was added at 50 μl per well as "inhibition column". And adding diluted serum with a series of concentrations into a titer column and a inhibition column according to the sequence of the concentrations, adding one titer hole and one inhibition hole for each concentration, wherein 50 mu L of each hole, and supplementing the total volume of the last two holes to 100 mu L by using PBS (phosphate buffer solution) to serve as a blank control group. After the above systems were mixed, incubated at 37℃for 30min, plates were washed 5 times with 20-fold dilution of wash solution PBST (0.01M PBS,0.05%Tween-20) and dried on absorbent paper.
(3) Adding enzyme-labeled secondary antibodies: anti-VHH-HRP (GenScript A01861-200) secondary antibody was diluted 5000-fold with PBST, 100. Mu.L was added to each well, incubated at 37℃for 30min, plates were washed 5 times with 20-fold dilution of wash solution PBST (0.01M PBS,0.05%Tween-20), and blotted dry on absorbent paper.
(4) TMB substrate chromogenic reaction and termination: equal volumes of TMB substrate solution A and B (Solarbio, PR 1210) were mixed in advance, 100. Mu.L was added to each well, incubated at 37℃for 10min, and 50. Mu.L was added to each well to terminate the reaction.
(5) Reading and data analysis: the absorbance at 450nm (OD 450 nm) was read with a microplate reader, the serum dilution factor corresponding to a titer column OD value between 1 and 1.5 was defined as "serum titer" (if there was more than one reading in this interval, a value close to 1 was taken), the inhibition well reading decrease corresponding to the titer well was defined as inhibition rate, and calculated by the following formula. According to the immune response result, lymphocytes with highest titer and inhibition rate are selected for preparing the nano antibody.
Figure BDA0003433342650000081
3. Isolation of Bactrian camel lymphocytes
Mixing Bactrian camel whole blood with equal volume of physiological saline at a volume ratio of 1:1 to obtain diluted blood, and standing at normal temperature. Into a sterile 50mL centrifuge tube, 20mL of lymph-separated liquid was added, and 20mL of diluted blood was slowly added along the tube wall with a sterile Pasteur pipette. Centrifuge 500g for 30min. The lymphocyte layer was taken into a new 50mL centrifuge tube, diluted 2-fold with physiological saline, centrifuged at 2000g for 10min at 4℃and the supernatant discarded. The lymphocytes were blown off with 5mL of physiological saline, centrifuged for 10min again at 2000g, and the supernatant was discarded to wash the lymphocytes thoroughly. Adding lysate (TRNSol) into each lymphocyte, subpackaging 1mL into 2mL centrifuge tube, and preserving at-80deg.C for use.
4. Extraction of Total RNA
The extraction of total RNA was performed according to Trizol reagent method of Invitrogen corporation. The specific method comprises the following steps:
the above lysate was added with 0.2mL of chloroform per 1 mL. The centrifuge tube was capped, vigorously shaken for 15 seconds, and incubated on ice for 5min. Centrifuge at 12000rpm for 10min at room temperature. Transferring the upper water phase with the volume not more than 80% to a new centrifuge tube, slowly adding 0.7 times of absolute ethyl alcohol, and uniformly mixing; transferring the obtained solution and the precipitate into GBC adsorption column, centrifuging at 12000rpm for 30 seconds, 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; 600. Mu.L Wash Buffer II,12000rpm, was added to the GBC column, centrifuged for 30 seconds, and the waste was discarded. Centrifuging at 12000rpm for 1min, discarding the waste liquid, opening the cover at room temperature, and air drying the residual rinse liquid in the adsorption column. Transferring GBC adsorption column into a new centrifuge tube, adding 30-100 mu L dd H 2 O, 2min at room temperature, 4℃and 12000 rpm. Collecting the liquid in the tube, and preserving at-80deg.C.
5. cDNA Synthesis
The first strand cDNA was synthesized using RNA as a template and referring to the instructions of Takara first strand reverse transcription kit. The specific method comprises the following steps:
(1) According to the first reaction system for cDNA synthesis shown in Table 1, the reagents were mixed in a centrifuge tube without nuclease and operated in an ice bath;
TABLE 1 first step reaction System for cDNA Synthesis
Total RNA 3μg
Oligo(dT) 18 primer 1μL
RNase free ddH 2 O Up to12μL
Total 12μL
(2) Incubating the reaction system at 65 ℃ for 5min, and cooling in an ice bath for 2min;
(3) Adding a reagent into the system after the previous step of reaction according to the second step of reaction system for cDNA synthesis shown in Table 2;
TABLE 2 second step reaction System for cDNA Synthesis
The system after the previous 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 performed at 42℃for 60min and at 70℃for 5min. The reverse transcription product cDNA was stored at-80 ℃.
6. 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 3 heavy chain antibody Gene primer sequences of Bactrian camels
Figure BDA0003433342650000101
The first round of PCR uses cDNA as its PCR template, and specific reaction parameters are shown in tables 4 and 5:
TABLE 4 nested PCR first-step reaction System
Figure BDA0003433342650000102
TABLE 5 first step reaction conditions for nested PCR
94℃ 5min
94℃ 30s
55℃ 30s
72℃ 1min,go to step2,30cycle
72℃ 10min
4℃ Forever
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, with the specific reaction parameters shown in tables 6 and 7
TABLE 6 nest type PCR second step reaction System
Figure BDA0003433342650000103
Figure BDA0003433342650000111
TABLE 7 nested PCR second step reaction conditions
Figure BDA0003433342650000112
7. Gene library construction
(1) Cleavage of the VHH target Gene and vector
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 (v/v) with the final concentration of 25%, taking 50 mu L of bacterial liquid, carrying out gradient dilution to determine the cell number, sub-packaging the rest bacterial liquid, and freezing at-80 ℃ to obtain the diazinon nanobody gene library.
8. Phage rescue
Inoculating cells with 10 times of reservoir capacity in 200mL LB (Amp) at 37 ℃ and culturing at 250rpm until OD600 is about 0.4-0.6; helper phage M13K07 (20:1 multiplicity of infection) was added, and after standing at 37℃for 30min, the mixture was incubated at 250rpm for 1h, kana antibiotic (1:1000) was added, and the mixture was incubated at 37℃overnight at 250 rpm. Centrifuging at 12000rpm for 15min at 4 ℃, taking supernatant, adding 1/5 volume of PEG/NaCl (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. Centrifuging at 12000rpm for 15min at 4 ℃, discarding the supernatant, re-suspending the precipitate with 1mL TBS, transferring to a 2mL centrifuge tube, centrifuging at 12000rpm for 5min at 4 ℃, filtering with a 0.22 μm polyethersulfone filter membrane, taking 10 μl of the obtained solution, adding 50% glycerol at final concentration, and preserving at-80 ℃.
2. Experimental results
The immunogen is used for the immunization of the Bactrian camel, and the detection antigen is used for the monitoring of immune response. The total time of the two-humped camel immunization is 5 times, the immune response situation is shown in figure 1, the serum titer of the two-humped camel is obviously improved from the three-immunity, the serum titer reaches 1:2000, and the inhibition rate is close to 80%; immune response has reached stability in four or five days, the potency is 1:4000, and the inhibition rate is about 89%. Thus, the fourth and fifth immunized blood is taken for pooling.
Example 3 affinity panning and identification of nanobodies
1. Experimental method
1. Affinity panning of nanobodies
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 oxazine farm artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mug/mL, adding the oxazine farm artificial antigen B-BSA into the micro-wells of the 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 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.
BSA was added to the diazinon nanobody phage library to a final BSA concentration of 1% (w/v), phage library (w/v) containing 1% BSA was added to 3 microwells with immobilized antigen, 100. Mu.L was added to each well, and incubated at 37℃for 1h. Unbound phage in wells were discarded, microwells were washed 10 times with PBST and then 5 times with PBS. Diazinon standard with the concentration of 5 mug/mL is added into each well, 100 mug/well is subjected to shake incubation for 4 hours at 37 ℃ to perform competition reaction, and liquid in the micropores is collected into a sterile centrifuge tube. The phage at this point is called "competing output", and the first round of screening is complete. Titers were determined from 10 μl of eluted phage, and the remaining E.coil TG1 strain grown to log phase for 4mL infection was amplified. The third day the amplified phage was precipitated with 5-fold dilution of PEG/NaCl solution (100 g PEG 8000 and 73.05g sodium chloride in water to 500 mL), and the phage titer was determined.
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
With coating liquid (0.375 g Na 2 CO 3 And 0.7325g NaHCO 3 Adding water to fix the volume to 250 mL), diluting the diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mug/mL, and standing at 4 ℃ overnight. 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, blocking was performed at 37℃for 2 hours, blocking solution was discarded, oven-dried at 37℃for 60 minutes, and stored at 4℃for use.
(2) Nanobody miniexpression
30 single colonies were randomly selected on the competition eluted output titer assay plates of the third and fourth panning screens, inoculated into 96-well plates with 0.5mL LB-Amp per well, and simultaneously inoculated with one single colony of e.coil TG1 not infected by phage as a negative control, and incubated overnight at 37 ℃ as a bacterial fluid "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
The deep well plate was centrifuged at 4000rpm for 20min, two ELISA plates with immobilized antigen were taken, 50. Mu.LPBS was added to each well of plate 1, PBS containing 10ng/mL of diazinon standard drug was added to plate 2, and the supernatant was aspirated from the centrifuged 96 well plate, and 50. Mu.L of enzyme-labeled wells were added to each well of the corresponding number. Incubation was carried out at 37℃for 30min, washed five times with 20-fold dilution of wash solution PBST (0.01 MPBS,0.05% Tween-20), and the intra-well liquid was dried. Anti-VHH-HRP (GenScript, A01861-200) was diluted 5000-fold with PBST, 100. Mu.L per well was added and incubated for 30min at 37 ℃. 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 developing solution (Solarbio, PR 1210) mixed with the same volume of color developing solution A and color developing solution B 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.
Figure BDA0003433342650000141
2. Experimental results
And sending phage clones 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 result shows that 1 diazinon nanobody is obtained and named NbEQ1.
NbEQ1 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 SEQ 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.
The amino acid sequence of VHH of the nano antibody NbEQ1 is shown in SEQ ID NO. 1:
EVQLLESGGGSVQAGSSLRLSCAGSGSNAPGVCVRWFRQTPGNDREWVATIDSTG YTAYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYCAARISWGLRVSCDGDFP YWGEGTQVTVSS；
the amino acid of the framework region FR1 of the nano antibody NbEQ1 is shown as 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 as SEQ ID NO. 4:
AYADSVKGRFTVSKEDAKRTVYLQMNRLRPEDTAMYYC；
the amino acid of the framework region FR4 of the nanobody is shown as SEQ ID NO. 5:
WGEGTQVTVSS；
the amino acid of the complementarity determining region CDR1 of the nanometer antibody NbEQ1 is shown in SEQ ID NO. 6:
GSNAPGVC；
The amino acid of the complementarity determining region CDR2 of the nanometer antibody NbEQ1 is shown in SEQ ID NO. 7:
IDSTGYT；
the amino acid of the CDR3 of the NbEQ1 complementarity determining region of the nanobody is shown in SEQ ID NO. 8:
AARISWGLRVSCDGDFPY。
meanwhile, the nucleotide sequence of the obtained gene for encoding the nano NbEQ1 is shown as SEQ ID NO. 9.
EXAMPLE 4 Mass production of nanobody NbEQ1
The preparation method of the nano antibody NbEQ1 in the form of protein expression comprises the following specific steps:
the obtained phage of the nano antibody NbEQ1 is cloned, the plasmid is extracted by a kit, and the plasmid is transferred into E.coil BL21 by a chemical conversion method. A single colony was picked from the transformation plate and inoculated into 10mL LB (Amp) medium, and incubated overnight at 37℃and 250 rpm. The overnight culture was incubated at 1:100 (v/v) in 750mL LB (Amp) medium, at 37℃and 250rpm until OD600 is about 0.4-0.6, IPTG was added to a working concentration of 1mM, at 37℃and 250rpm, and the culture was continued overnight. Centrifuging at 12000rpm for 5min at 4deg.C the next day, collecting bacterial precipitate, centrifuging at 12000rpm for 10min by sucrose osmotic pressure freeze thawing method, collecting supernatant, and subjecting the supernatant to affinity chromatography purification to obtain nanometer antibody NbEQ1 (amino acid sequence shown in SEQ ID NO. 1) shown in figure 2.
EXAMPLE 5 determination of working concentration and sensitivity of nanobody NbEQ1
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 diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mug/mL, adding the diluted diazinon artificial antigen B-BSA into 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 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
The purified nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 is diluted by PBS to perform two-time gradient dilution, so as to obtain a series of nano antibodies with different concentrations. The ELISA plate was added with 50. Mu.L of PBS per well, followed by 50. Mu.L of antibody after gradient dilution, and 3 wells of replicates were performed for each antibody concentration, while a blank (100. Mu.L of PBS) was run with 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), 100. Mu.L of Anti-VHH-HRP (GenScript, A01861-200) secondary antibody diluted 5000-fold with PBST was added to each well after drying on absorbent paper. Incubating at 37deg.C for 30min, washing the plate with 20-fold diluted PBST (0.01M PBS,0.05%Tween-20) solution for 5 times, adding 100 μl of TMB color developing solution into each well after drying on absorbent paper, incubating at 37deg.C in dark for 10min, and adding 50 μl of stop solution into each well 10％H 2 SO 4 (v/v) the OD at 450nm was read on a microplate reader.
The antibody concentration of OD450nm between 1 and 1.5 is the working concentration of the nano antibody, and the working concentration of NbEQ1 is 91.5ng/mL under the experimental conditions.
3. Drawing standard curve by indirect competition ELISA
Diazinon standards were diluted with PBS to give a series of diazinon solutions of different concentrations, added to the elisa plate, 50 μl per well, 3-well replicates were run for each concentration, and a 3-well drug blank (50 μl PBS) was prepared. The nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 is diluted to the working concentration of 91.5ng/mL by PBS, and 50 mu L of diluted antibody is added to each hole. 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), 100. Mu.L of Anti-VHH-HRP (GenScript, A01861-200) secondary antibody diluted 5000-fold with PBST was added to each well after drying on absorbent paper. Incubation at 37deg.C for 30min, washing the plate with 20-fold diluted PBST (0.01M PBS,0.05%Tween-20) solution for 5 times, adding 100 μl of TMB color developing solution into each well after drying on absorbent paper, incubating at 37deg.C in dark for 10min, and 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 OD450 values of drug blank group was recorded as B 0 The average OD450 values at different drug concentrations were reported 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.
2. Experimental results
An indirect competition ELISA standard curve established based on an antibody NbEQ1 with an amino acid sequence shown in SEQ ID NO.1 is shown in FIG. 3, and the standard curve is of an S type, has good linear correlation, the detection range is 2.154 ng/mL-12.031 ng/mL, the IC50 is 5.091ng/mL, the minimum detection Limit (LOD) is 0.161ng/mL, and can meet the detection requirement of 0.01mg/kg of the maximum residual limit of China.
Example 6A method for detecting diazinon
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 diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mug/mL, adding the diluted diazinon artificial antigen B-BSA into 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 the mixture was allowed to stand at 37℃for 2 hours, and the liquid in the well was poured out, and the mixture was dried on absorbent paper and baked at 37℃for 1 hour, and then stored at 4℃for use.
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 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 NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 was diluted to a working concentration of 91.5ng/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. Washing the microwells with 20-fold diluted wash solution PBST (0.01M PBS,0.05%Tween-20) 5 times, and drying 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, 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 developing solution previously mixed with an equal volume of color developing solution A and color developing solution B (Solarbio, PR 1210) 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 The Bx/B0 ratios for different drug concentrations or sample wells were calculated and standard deviations for each set of parallel data. To be used forLogarithmic value of standard substance concentration is abscissa, B x /B 0 The ratio is the ordinate, and a standard curve graph is drawn. And 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 diazinon concentration, and the anti-logarithmic value is obtained, namely the diazinon 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 7 diazinon detection kit
1. Composition of the kit
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 diazinon artificial antigen B-BSA shown in the structural formula (IV) prepared in the example 1 to 1 mug/mL, adding the diluted diazinon artificial antigen B-BSA into 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 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 the absorbent paper and drying the liquid 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: diazinon standard solutions of different concentrations.
4. Dilution of antibody with sample: PBS.
5. Dilutions of labeled antibodies: PBST.
6. Developing A solution and B solution.
7. Stop solution: 10% H 2 SO 4 (v/v)。
8. Concentrated washes (20×): PBST (0.01M PBS,0.05%Tween-20), after 20-fold dilution, was used for plate washing.
9. Detection of antibodies: the amino acid sequence of the nanometer antibody NbEQ1 is shown as 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 Add 50. Mu.L PBS to each wellInto each well, 50. Mu.L of the diluted sample solution was added to 50. Mu.L of the standard solutions of different concentrations. The nanobody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 was diluted to a working concentration of 91.5ng/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 dilution of 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 micropores for 5 times by using a washing solution PBST (0.01M PBS,0.05%Tween-20) diluted by 20 times, beating to dryness on absorbent paper, adding 100 mu L of TMB developing solution which is prepared by mixing an equal volume of developing A solution and developing B solution in advance into each hole, incubating at 37 ℃ for 10min in a dark place, adding 50 mu L of stop solution into each hole, 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 Calculation of B for different drug concentrations or sample wells with Excel 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. And 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 diazinon concentration, and the anti-logarithmic value is obtained, namely the diazinon 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 NbEQ1
1. Experimental method
Other 12 diazinon analogues (methylpyrimidinone, diazoxon, aqueous amifos, quiniphos, fenitrothion methyl parathion, ethyl parathion, methidathion, triazophos, phoxim, profenofos) and diazinon metabolites (IMHP) were tested using the kit of example 7.
Standard solutions of various diazinon analogs were prepared, and standard curves for each diazinon analog and metabolite were plotted to obtain respective IC50 values according to the various diazinon analogs detected in gradient concentrations using the kit of example 7. The cross-reactivity of each drug with nanobody NbEQ1 was calculated using the following formula:
Figure BDA0003433342650000181
2. Experimental results
The results are shown in Table 8, the cross reaction rates of the nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1, diazinon analogues of methyl pyrimidine phosphorus and diazinon are 6.877% and 1.324%, respectively, and the cross reaction rates of the nano antibody NbEQ1 with other analogues are lower than 1% (the IC50 is greater than 500 ng/mL), which indicates that the nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 can specifically identify diazinon, and the established detection method and the detection kit have higher specificity for diazinon detection.
TABLE 8 sensitivity and specificity of diazinon nanobody NbEQ1
Figure BDA0003433342650000191
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Figure BDA0003433342650000201
Example 9 Activity of nanobody NbEQ1 in organic solvents at different concentrations
1. Experimental method
The nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 is diluted to the working concentration of 91.5ng/mL by PBS. Different volume ratios (0%, 20%, 40%, 60%, 80%, 100%) of methanol, acetonitrile, acetone solutions were prepared with PBS, respectively. 50 mu L of solution prepared by organic solvents with different volume ratios is added into each hole, and then 50 mu L of diluted antibody solution is added. The binding ability of the antibody to the detection antigen under the condition of different organic solvents was measured by using the detection kit in example 7, and the tolerance ability of the nanobody NbEQ1 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. Normalized data were performed with the titer at 0 for the organic solvent as 100%. The measurement result is shown in fig. 4, and the binding activity of the nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 is reduced along with the increase of the concentration of the organic solvent, especially the reduction trend in the organic solvent of acetonitrile is the fastest. At an acetonitrile concentration of 10%, nanobody NbEQ1 has lost 60% of the 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 deactivated. The nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 has poor tolerance to three common organic solvents such as methanol, acetonitrile and acetone in pretreatment of a standard method, especially can not tolerate acetonitrile, and the organic solvent should be removed in an antibody working solution as much as possible, so that the influence of the high-concentration organic solvent on the sensitivity of the antibody is avoided.
Example 10 Activity of nanobody NbEQ1 at different temperatures
1. Experimental method
The nano antibody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 is diluted to the working concentration (91.5 ng/mL) by PBS and then incubated for different times (0, 10, 20, 30, 40, 50 and 60 min) at the high temperature of 85 ℃. Binding activity of the antibodies to the test antigen after various times of high temperature treatment was measured using the kit of example 7, respectively.
2. Experimental results
Normalized data were performed with a titer at a heating time of 0 as 100%. The measurement result is shown in fig. 5, and the binding activity of the nanobody NbEQ1 with the amino acid sequence shown as SEQ ID NO.1 and the detection antigen binding activity is reduced along with the prolonged incubation time. The nanobody 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 nano antibody NbEQ1 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 NbEQ1 to have great application advantages in a relatively high-temperature detection environment.
Example 11 Activity of nanobody NbEQ1 at different pH conditions
1. Experimental method
The solvent of the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 was diluted to a working concentration (91.5 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, 10.4) as a diluent. The binding ability of the antibody to the detection antigen was measured using the kit of example 7, and the binding activity of the nanobody NbEQ1 having the amino acid sequence shown in SEQ ID NO.1 under different pH conditions was evaluated.
2. Experimental results
Normalized data were taken as 100% at a pH of 7.4. As shown in FIG. 6, the antigen binding activity of the nano antibody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 is gradually reduced in the pH range of 3.4-10.4, and the antigen binding activity is higher under acidic conditions. When the pH was lowered to 2.4, the antibody was substantially inactivated. In general trend, nanobody NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 can maintain better antigen binding activity in a wider pH range, but is more suitable for working in an acidic environment with pH not lower than 3.4.
EXAMPLE 12 storage stability of nanobody NbEQ1 under different temperature conditions
1. Experimental method
Adding 0.03% preservative ProClin 300 (v/v) and 0.01% protease inhibitor (v/v) into nano antibody NbEQ1 solution with amino acid sequence shown in SEQ ID NO.1, 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 NbEQ1 with the amino acid sequence shown in SEQ ID NO.1 to the detection antigen 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 storage stability of the antibody after the preservative and the protease inhibitor are added is detected so as to determine the shelf life of the nano antibody with the amino acid sequence shown as SEQ ID NO. 1. Normalized data processing was performed with the titer at 0 day of storage time as 100%. As shown in fig. 7, after the nanobody NbEQ1 with the base acid sequence shown in SEQ ID No.1 is stored at-20 ℃ and 4 ℃ for two months, the antigen binding activities of the nanobody NbEQ1 and the detection antigen are 62%, 50% and 60% respectively, and even the nanobody NbEQ1 with the amino acid sequence shown in SEQ ID No.1 is stored at a higher temperature (37 ℃) for about half a month, the antigen binding activity of the nanobody NbEQ1 is 37%, and the storage time at 37 ℃ is more than one month, the titer is not reduced but the titer is increased, which is probably caused by the fact that the concentration of the antibody solution is increased due to the evaporation of the antibody solution. In summary, nanobody NbEQ1 has a certain storage stability, can be transported, stored and used for a long time at room temperature after adding preservative or protease inhibitor, and can be stored even in a short time at a relatively high temperature of 37 ℃.
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> a nanobody for detecting diazinon and application thereof
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<170> SIPOSequenceListing 1.0
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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Ser
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Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Ser Asn Ala Pro Gly Val
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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
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Ala Arg Ile Ser Trp Gly Leu Arg Val Ser Cys Asp Gly Asp Phe Pro
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Tyr Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser
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Ser Leu Arg Leu Ser Cys Ala Gly Ser
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Val Arg Trp Phe Arg Gln Thr Pro Gly Asn Asp Arg Glu Trp Val Ala
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Thr
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Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Lys Glu Asp
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Thr Ala Met Tyr Tyr Cys
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Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser
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Pro Tyr
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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 (10)

1. A nano antibody for specifically recognizing diazinon 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 diazinon 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 coding 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 encoding gene of claim 2, the recombinant vector of claim 3, and/or the recombinant cell of claim 4 for preparing an immunological detection kit of diazinon.
6. A method for the detection of diazinon for non-diagnostic purposes, characterized in that the nanobody of claim 1 is used.
7. The method according to claim 6, wherein the detection is performed by an indirect ELISA method, wherein the diazinon hapten represented by formula (II) is coupled to a carrier protein to obtain a diazinon complete antigen as a detection antigen, wherein the nanobody is used as a detection antibody,
Figure FDA0004171090300000011
8. a kit for detecting diazinon comprising the nanobody of claim 1.
9. The kit of claim 8, wherein the nanobody is coated on a solid carrier, and further comprises a diazinon complete antigen obtained by coupling the diazinon hapten with a carrier protein as a detection antigen in the formula (II),
Figure FDA0004171090300000012
Figure FDA0004171090300000021
10. the kit of claim 8, further comprising an enzyme-labeled secondary antibody, a chromogenic agent, and a terminator.
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CN108794632A (en) * 2017-12-28 2018-11-13 华南农业大学 A kind of nano antibody and enzyme-linked immune analytic method of Broadspectrum specificity identification diethoxy organophosphorus pesticide
CN110407943A (en) * 2019-04-25 2019-11-05 华南农业大学 A kind of nano antibody and its preparation method and application of carbofuran pesticide

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108794632A (en) * 2017-12-28 2018-11-13 华南农业大学 A kind of nano antibody and enzyme-linked immune analytic method of Broadspectrum specificity identification diethoxy organophosphorus pesticide
CN110407943A (en) * 2019-04-25 2019-11-05 华南农业大学 A kind of nano antibody and its preparation method and application of carbofuran pesticide

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