CN113388037A - Preparation and application of specific recognition fenitrothion nano antibody - Google Patents
Preparation and application of specific recognition fenitrothion nano antibody Download PDFInfo
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Abstract
The invention discloses a preparation method and application of a specific recognition fenitrothion nano antibody. The amino acid sequence of the nano antibody is shown in SEQ ID NO. 1; the nucleotide sequence of the gene for coding the nano antibody is shown as SEQ ID NO. 9. The nano antibody disclosed by the invention can be applied to the detection of practical samples of fenitrothion residues, can specifically detect fenitrothion, has the characteristics of higher thermal stability, organic tolerance, weak acid resistance and the like, can improve the sensitivity of fenitrothion immunodetection, and is simple to operate and short in consumed time. 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 high application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to preparation and application of a specific recognition fenitrothion nano antibody.
Background
Fenitrothion (Fenitrothion), also known as Fenitrothion, is a widely used organophosphorus pesticide. Fenitrothion has better contact poisoning and stomach poisoning effects, can control hemiptera, coleopteran and other pests, and is commonly used for controlling the pests on corn, rice, soybean and vegetables. Fenitrothion has moderate toxicity, and unreasonable application of fenitrothion can cause residual accumulation of fenitrothion on crops, and excessive intake of fenitrothion has certain toxic action on human and livestock: resulting in accumulation of single bonds in the nervous system, resulting in symptoms such as nausea, vomiting, abdominal pain, diarrhea, coma, and acute poisoning which can also endanger life.
Accurate detection of fenitrothion remained in crops is an important means for avoiding the injury of fenitrothion to human and livestock health. At present, the detection method of fenitrothion in the national standard is still an instrument analysis method, such as a detection method combining chromatography and mass spectrum, and the like. Such detection methods have high sensitivity and accuracy, but require expensive equipment and reagents and specialized operators, and in addition, require complicated sample preparation processes, which are time consuming and cost prohibitive to meet the needs of the current market for screening large quantities of agricultural products.
The immunoassay method is an analysis method which is developed in recent years and is based on antibody specificity to recognize antigen molecules and reflect the content of a substance to be detected through the signal intensity released by signal molecules. The immunoassay method has the characteristics of capability of specifically identifying one or a class of target analytes, high sensitivity, simple equipment, low professional requirement on operators and short detection time compared with an instrumental analysis method, and can perform semi-quantitative or quantitative analysis on a sample to be detected even within a few minutes by some immunoassay methods. Commonly used immunoassay methods include enzyme-linked immunoassay, lateral flow immunoassay, fluorescence immunoassay, electrochemical immunoassay, and the like. The key to the success of immunoassay methods is the production of antibodies with high sensitivity and specificity. Since antibodies are proteins in nature, immunoassays must be carried out under mild conditions (saline ion solution of appropriate concentration, environment near 37 ℃, pH value that is more neutral, organic solvent of low concentration, etc.), and it is necessary to avoid inactivation of the immunological formulation during storage and transportation, affecting its detection activity.
The nano antibody is a novel antibody preparation. Heavy chain antibodies that naturally lack a light chain were found in llama by belgium scientists in 1993. Later, people express the antigen binding region of the heavy chain antibody through a 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 a genetic engineering means at present, the molecular weight of the heavy chain antibody fragment is only about 15kD, which is only about one tenth of that of the traditional antibody, and the size of the heavy chain antibody fragment is in a nanometer level, so the heavy chain antibody fragment is also called as a nano antibody.
In the process of application of the nano antibody, people find that the nano antibody has properties different from those of the traditional antibody, wherein the most typical property is that the nano antibody can still retain higher antigen binding capacity after high-temperature treatment and in organic solution with certain concentration, and the properties can simplify the pretreatment process of a sample to a certain extent, so that the immunoassay method can better meet the requirement of the market on quick detection.
Chinese patent: the fenitrothion hapten, the artificial antigen, the specific antibody and the application (CN101012239B) thereof provide a technical scheme for detecting fenitrothion by utilizing the artificial fenitrothion antibody through an indirect ELISA method and a direct ELISA method. As can be known from the specification of the Chinese patent, when the fenitrothion is detected by an indirect ELISA method, IC50 is 13 mug/L, and the ultrahigh-sensitivity detection method for fenitrothion is not provided at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a fenitrothion nano antibody and application thereof.
The first purpose of the invention is to provide a nano antibody sm6 for anti-fenitrothion.
The second purpose of the invention is to provide a gene for coding the nano antibody sm 6.
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 gene, the recombinant vector or the recombinant cell in the immunological detection analysis for detecting fenitrothion.
The sixth purpose of the invention is to provide a fenitrothion detection method.
The seventh purpose of the invention is to provide a reagent kit for detecting fenitrothion.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention constructs an alpaca immune antibody library, coats a detection antigen solid phase on an enzyme label plate by using a phage display technology, and puts the alpaca immune antibody library into the enzyme label plate for affinity panning to obtain a nano antibody specifically combined with fenitrothion, wherein the nano antibody has an amino acid sequence shown in SEQ ID NO. 1. The method is applied to the immunological detection and analysis of fenitrothion, and a rapid, sensitive and stable detection method of fenitrothion is established through the immunological detection and analysis.
Accordingly, the invention claims the following:
a specific recognition fenitrothion and nano antibody sm6, which has an amino acid sequence shown in SEQ ID NO. 1; the framework regions (FR1-FR4) of the amino acid sequence of the nano antibody are respectively selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the complementarity determining regions (CDR1-CDR3) are respectively selected from SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO. 8.
The gene for coding the specific recognition fenitrothion nano antibody has a nucleotide sequence shown in SEQ ID No. 9.
A recombinant vector comprising the nucleotide sequence of the gene of the second object of the present invention.
Preferably, the vector is an expression vector.
Preferably, the expression vector is a pComb3X phagemid vector carrying a nano antibody sm6 gene.
A recombinant cell comprising the recombinant vector according to the third object of the present invention.
Preferably, the cell is an E.coli cell.
The application of one or more of the nano antibody, the gene, the recombinant vector or the recombinant cell in the immunological detection and analysis for detecting fenitrothion.
A fenitrothion detection method, which utilizes the anti-fenitrothion nano antibody.
Preferably, the detection method is based on an indirect ELISA method, the fenitrothion antigen shown in the formula (I) is used as a detection antigen,
preferably, the carrier protein of the fenitrothion complete antigen is selected from one or more of bovine serum albumin, keyhole limpet hemocyanin or lactoferrin.
More preferably, the carrier protein is Bovine Serum Albumin (BSA).
A kit for detecting fenitrothion utilizes the anti-fenitrothion nano antibody.
Preferably, the detection kit is used for detection based on an indirect ELISA method, and also comprises a fenitrothion complete antigen shown in formula (I) as a detection antigen,
preferably, the carrier protein of the fenitrothion complete antigen is selected from one of bovine serum albumin, keyhole limpet hemocyanin and lactoferrin.
Most preferably, the carrier protein is Bovine Serum Albumin (BSA).
Preferably, the detection kit further comprises an enzyme-labeled antibody, a color developing agent and a stop solution.
Enzyme-labeled antibody: HRP (horse radish peroxidase) -labeled anti-HA secondary antibody;
color developing agent: TMB color development liquid;
stopping liquid: 10% H2SO4(v/v)。
More preferably, the use method of the detection kit is as follows:
s1 test antigen B-BSA is diluted to 0.5. mu.g/ml by PBS, 0.1ml is added to each well of an enzyme label plate, the temperature is kept overnight at 4 ℃, after the next day, PBST is used for washing twice, PBS is used for preparing skimmed milk powder (w/v) with 2 percent in situ as a blocking solution, 150. mu.L is added to each well, the blocking solution is sealed for 1h at 37 ℃, the blocking solution is discarded, and PBST is used for washing twice. Oven drying at 37 deg.C for 30min, and storing at 4 deg.C;
s2, adding a sample to be tested (blank, negative and positive hole controls are simultaneously made) into the enzyme label plate, adding 50 mu L of antibody into each hole, diluting the fenitrothion-resistant nano antibody to a working concentration (13ng/mL) by PBS, adding 50 mu L of antibody diluent into each hole, incubating for 30min at 37 ℃, and washing the plate for 5 times by PBST;
s3 adding 100 mu L of HRP (horse radish peroxidase) -labeled anti-HA secondary antibody diluted 5000 times by PBST (Poly-p-phenylene benzobisoxazole) into each hole after patting dry on absorbent paper, incubating for 30min at 37 ℃, washing the plate for 5 times by PBST, and patting dry the liquid in the hole;
s4 adding 100 μ L of TMB color developing solution into each well, incubating for 10min at 37 deg.C in dark;
s5 Add 50. mu.L 10% H per well2SO4(v/v) stop solution, and reading the OD value of 450nm on a microplate reader.
The nano antibody can be prepared in a large scale by a gene engineering recombination expression mode, wherein the gene for coding the nano antibody is cloned to an expression vector to be prepared in a large scale in a protein expression mode.
After the nano antibody is expressed by prokaryotes, immunological detection and analysis are carried out in a protein form.
Compared with the prior art, the invention has the following beneficial effects:
the method comprises the steps of immunizing alpaca, establishing an antibody gene library by using immunized alpaca lymphocytes, wherein the antibody gene library has good gene diversity of nano antibodies, and screening the antibody gene library to obtain the nano antibody aiming at fenitrothion, wherein the nano antibody can be applied to the detection of practical samples of fenitrothion residues. The nano antibody has the characteristics of higher thermal stability, higher organic tolerance, weak acid resistance and the like, can improve the sensitivity of fenitrothion immunodetection, and has the advantages of simple operation and shorter time consumption. 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 high application value.
Drawings
FIG. 1 is the structural diagram of fenitrothion nano antibody hapten and complete antigen.
FIG. 2 is a diagram of nano antibody sm6 SDS-PAGE.
FIG. 3 is a standard curve of indirect competition ELISA based on nanobody (Nbsm 6).
Fig. 4 is a schematic diagram of the activity of the nanobody sm6 at different concentrations of acetone and acetonitrile.
Fig. 5 is a schematic diagram of the activity of the nanobody sm6 at different temperatures.
Fig. 6 is a diagram showing the activity of the nanobody sm6 in PBS with different pH.
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 construction of an alpaca immune antibody library
1. Preparation of immune antigen and detection antigen
The design and synthesis of fenitrothion hapten are completed by early work in a laboratory, and the experiment uses two haptens with structures in total. The hapten structure A is used for preparing artificial immune antigen, and the hapten structure B is used for preparing artificial detection antigen. The structures of the hapten and the artificial antigen are shown in figure 1.
Hapten A and Lactoferrin (Lactoferrin, LF) are coupled through an active ester method to form immune antigen. The specific operation method comprises the following steps: 7.3mg EDC and 4.3mg NHS were weighed and 0.1mL DMF was added to dissolve the solids. And adding 9.7mg of fenitrothion hapten A into the mixture solution, and stirring at room temperature in the dark or carrying out shake reaction for 4 hours to activate the hapten. 50mg of LF was weighed and added to 5mL of carbonate buffer (pH 9.4) to prepare a 10mg/mL protein solution. The activated hapten was added dropwise with stirring, and the solution was stirred overnight at room temperature with pH paper to determine the alkaline range. Dialyzing with PBS for 3 times, subpackaging, and freezing at-20 deg.C. The coupled fenitrothion A-LF is used as an immune antigen.
Hapten B is coupled with Bovine Serum Albumin (BSA) to form detection antigen B-BSA. The specific operation method comprises the following steps: 26.9mg of fenitrothion hapten B is weighed and added with 0.5mL of 1,4 dioxane for dissolution. 200mg of BSA was weighed and added to 20mL of carbonate buffer (pH 9.4) to prepare a 10mg/mL protein solution. The activated hapten was added dropwise with stirring, and the solution was stirred overnight at room temperature with pH paper to determine the alkaline range. Dialyzing with PBS for 3 times, subpackaging, and freezing at-20 deg.C. The coupled fenitrothion B-BSA is used as a detection antigen.
2. Alpaca immunization protocol
Animal immunization is carried out on healthy alpaca, fenitrothion A-LF is used as an immune antigen, subcutaneous injection is carried out on the back neck of the alpaca, and the immune dose is 0.5mg of the immune antigen each time. The first immunization is carried out by mixing and emulsifying 0.5mL of complete Freund's adjuvant and immunizing antigen, the subsequent boosting immunization is carried out by emulsifying 0.5mL of incomplete Freund's adjuvant and antigen, and 3 boosting immunizations are carried out at 2 weeks intervals.
Serum was isolated in 10mL of blood before immunization as a negative control. From the second immunization, 10mL of blood after one week of each immunization was taken for serum titer and competition reaction detection. And after the third immunization and the fourth immunization, collecting 50-100 mL of peripheral blood for constructing a nano antibody library.
3. Isolation of alpaca lymphocytes
The method for collecting alpaca peripheral blood needs to separate lymphocytes as soon as possible, and comprises the following specific operation methods: the alpaca peripheral blood and sterile physiological saline are mixed and diluted in equal volume in a clean container without RNA enzyme. The diluted peripheral blood was centrifuged with a commercial lymphocyte separation medium, and blood cells of different densities were centrifuged to distribute the lymphocyte separation medium at different depths, wherein the lymphocytes formed a white cell layer at a depth of about 1/3 below the surface of the liquid. Because the density of the lymphocyte separation liquid is influenced by the temperature and the performance of centrifuges in different laboratories is different, the optimal separation conditions need to be found out by self according to the instruction of the specification, and the reference centrifugation parameters are as follows: room temperature (about 25 ℃); 500 g; and (3) 30 min. The success rate can be improved by adopting fresh blood for separation, reducing the volume of each centrifugation, using a centrifugal tube special for separating lymphocytes and other measures. After lymphocyte separation, carefully collecting the lymphocyte separated liquid in an enzyme-inactivating clean container by using a rubber-head dropper or a pipette without RNase, washing the redundant lymphocyte separated liquid on the cell surface by using sterile normal saline, and centrifugally collecting cell precipitates. Adding lysis solution TRNsol into the collected lymphocytes, wherein 1-2 mL TRNsol is needed for the lymphocytes separated from 10mL of blood, repeatedly blowing and beating the lymphocytes by using a pipette or a rubber head dropper without RNase until the lymphocytes are completely lysed in the TRNsol, and storing the TRNsol at-80 ℃ for later use for at least one year.
4. Extraction of Total RNA
Extracting total RNA from the lymphocytes preserved in TRNsol lysate by using a commercial RNA extraction kit or other effective RNA extraction methods, and operating according to the instruction.
After extraction of total RNA, a small sample was subjected to nucleic acid electrophoresis and the RNA concentration was determined in a ultramicrospectrophotometer (nanodrop). The ideal RNA sample should be intact without degradation, clear 28S and 18S bands are visible from the nucleic acid electrophoresis gel, no genomic DNA is mixed, and the ratio of the ultraviolet absorbance values at 260nm and 280nm (A260/280) should be around 2.0. If genomic DNA contamination occurs, the genomic DNA should be removed by DNase before reverse transcription, and again the genomic DNA is removed and RNA is not degraded in the process as verified by electrophoresis; if the RNA has been degraded, it is necessary to re-extract the RNA. RNA should be reverse transcribed into cDNA as soon as possible or stored briefly in an environment at-80 ℃.
5. Synthesis of cDNA
And (3) carrying out reverse transcription on mRNA in the RNA sample obtained in the last step into cDNA by adopting a commercial reverse transcription kit, and operating according to the instruction. After the reverse transcription is finished, the products are firstly mixed uniformly, then are packaged in different sterile centrifuge tubes, and are stored in an environment of minus 80 ℃.
6. Amplification of target gene of nano antibody
The target gene is amplified by two steps by adopting nested PCR, and the sequences of the primers are shown in a table 1.
TABLE 1 alpaca heavy chain antibody Gene primer sequences
The first round of PCR uses cDNA as a first round of PCR template, and the specific reaction parameters are shown in Table 2:
TABLE 2 nested PCR first step reaction System and reaction conditions
The PCR product of the first step can generate two product bands of 1000bp and 750bp after nucleic acid electrophoresis, the 750bp band is cut and recovered, and the concentration is measured.
Second round PCR the second round PCR amplification was performed using the recovered product of the first round PCR as a template, and since there are two subtypes of the light chain and heavy chain antibody of the alpaca (IgG2, IgG3), different primer pairs were required for the second round amplification. F and R1 are used for amplification of an IgG2 gene, and F and R2 are used for amplification of an IgG3 gene.
TABLE 3 nested PCR second step reaction System and 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 to the final concentration, taking 50 mu L of bacterial liquid, performing gradient dilution to determine the cell number, subpackaging the rest bacterial liquid, and freezing and storing at-80 ℃. Namely the fenitrothion nano antibody gene bank.
8. Phage rescue
Inoculating cells with more than 10 times of library capacity into 200mL LB-Amp according to the cell number measuring result of the transgenic escherichia coli, and culturing at 37 ℃ and 250rpm until logarithmic phase; 1mL of the mixture was added to the flask with a titer of 1012The helper phage M13K07 was left to stand at 37 ℃ for 30min at cfu/mL or more, and then cultured at 250rpm for 1h, and kanamycin (50. mu.g/mL) was added thereto and cultured at 37 ℃ overnight at 250 rpm. Centrifuging at 12000rpm and 4 ℃ for 15min, taking the supernatant, adding 1/4 volumes of PEG/NaCl, and carrying out ice bath for 2-3 h. Centrifugation was carried out at 12000rpm for 15min at 4 ℃ and the supernatant was discarded, the pellet was resuspended in 1mL TBS, transferred to a 2mL centrifuge tube, centrifuged at 12000rpm for 5min at 4 ℃ and filtered through a 0.22 μm polyethersulfone filter. And (3) taking 10 mu L of phage for measuring titer, adding glycerol with the final concentration of 50 percent into the rest, and preserving at minus 80 ℃ to obtain the fenitrothion nano antibody phage library.
Example 2 affinity panning and identification of Nanobodies
First, experiment method
1. Affinity panning of Nanobodies
(1) Detection of antigen immobilization
The detection antigen B-BSA was diluted to 1. mu.g/mL with PBS, added to wells of an ELISA plate at 100. mu.L per well, and allowed to stand overnight at 4 ℃. The following day, after washing the plates twice with PBST (0.01M PBS, 0.05% Tween-20), 150. mu.L of 1% BSA-PBS (w/v) solution was added to each well and allowed to stand at 37 ℃ for 1 h. The liquid in the hole is poured out and patted dry on absorbent paper for later use.
(2) Positive phage selection
BSA was added to the phage library of example 1 to give a final BSA concentration of 1% (w/v), and the mixture was added to 3 wells containing immobilized antigen, 100. mu.L per well, and incubated at 37 ℃ for 1 h. Free phage in wells were discarded, wells were washed 10 times with PBST and 5 times with PBS. Gly-HCl (0.1M, pH 2.2) was added and acid eluted at 37 ℃ for 10min, immediately neutralized with 1M Tris solution and the above procedure was repeated for a second acid elution.
And combining the two acid washing and removing solutions, adding a fenitrothion standard substance to a final concentration of 1 mu g/mL, standing at 37 ℃ for 30min for competitive reaction, and collecting the liquid in the micropores into a sterile centrifuge tube. The phage at this point is called "output" and the first round of screening is complete.
The phage titer is determined before and after screening, and the specific operation method is as follows:
10 μ L phage was diluted in 10-fold gradient, and the expected titer and two dilutions in tandem (typically 10-fold input dilution) were selected7、108、109The output dilution factor is 102、103、104) 10 mul of diluent is taken to infect E.coil TG1 in log phase of 100 mul, and is kept still for 30min at 37 ℃, an LB-Agar-Amp plate is coated, and colony counting and phage titer calculation are carried out after overnight culture, and the calculation formula is as follows:
(3) phage amplification
After the screened phage is amplified, the phage is used for the next screening, and the method for amplifying the phage is as follows:
half of the output phage was added to 4mL log phase E.coil TG1 and left for 30 min. LB medium was added to a total volume of 10mL and Amp was added to working concentration (10. mu.g/mL), shaken at 37 ℃ 250rpm for 30min, 50. mu.L was added to a titer of 1012cfu/mL M13KO7 helper phage, standing for 30min, shaking at 37 ℃ 250rpm for 1h, adding LB to a total volume of 100mL, adding Amp and Kana (kanamycin ) working concentration (Kana working concentration is 50. mu.g/mL), and shaking at 37 ℃ 250rpm overnight.
And (3) centrifuging the bacterial liquid, transferring the supernatant into a new centrifugal bottle, adding 1/4-volume PEG/NaCl solution, mixing uniformly, carrying out ice bath for 2h, centrifuging at 12000rpm for 20min, discarding the supernatant, reversing the supernatant on absorbent paper to remove residual moisture as much as possible, resuspending the white precipitate by using 1mL PBS containing 1% BSA, filtering the white precipitate by using a 0.22-micron polyethersulfone filter membrane, storing the product at-20 ℃ to serve as the next round of screening input, and repeating the screening scheme in the step (2).
The concentration of immobilized antigen B-BSA in the above screening steps is respectively reduced to 1. mu.g/mL, 400ng/mL and 100ng/mL in 4 rounds, and the concentration of the drug used in the competitive reaction is respectively reduced to 100ng/mL, 10ng/mL and 1ng/mL in 2 nd, 3 th and 4 th rounds. The conditions can be adjusted according to the actual immunity condition, and if the serum titer is lower, the concentration of the detection antigen B-BSA during the panning can be properly improved; if the inhibition rate is low, the concentration of the drug in the competitive reaction needs to be increased appropriately.
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
The detection antigen B-BSA was diluted to 1. mu.g/mL with PBS and left to stand overnight at 4 ℃. After washing twice with PBST the next day, 2% skimmed milk powder (w/v) was prepared in situ with PBS as blocking solution, 150. mu.L of each well was added, blocking was performed at 37 ℃ for 1h, the blocking solution was discarded, and washing was performed twice with PBST. Oven drying at 37 deg.C for 30min, and storing at 4 deg.C for use.
(2) Nanobody miniexpression
And randomly selecting 96 single colonies on an output titer determination plate of the third round of panning and the fourth round of panning, inoculating the single colonies into a 96-well plate containing 0.5mL of LB-Amp in each well, simultaneously inoculating one E.coil TG1 single colony which is not infected by the phage as a negative control, and culturing at 37 ℃ overnight to be used as a bacterial liquid 'mother plate'.
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 containing 1mL of LB-Amp in each hole, culturing the inoculated holes corresponding to the mother plate at 37 ℃ and 180rpm for 3h, adding IPTG (working concentration of 1mM) into each hole, culturing the holes at 37 ℃ and 180rpm overnight. The master plate was stored at 4 ℃ until use.
(3) Enzyme-linked immunoassay positive clone
Centrifuging the deep-well plate at 4000rpm for 20min, taking two enzyme-labeled plates with immobilized antigens, adding 50 mu L of PBS into each hole of the plate 1, adding PBS containing 1 mu g/mL fenitrothion standard drug 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 30min at 37 ℃, wash five times with PBST, and pat dry the liquid in the wells. Since the expressed antibody carries a 6 His-tag and HA-tag, Anti-HA-HRP secondary antibody was diluted 5000-fold with PBST, 100. mu.L was added per well, and incubated at 37 ℃ for 30 min. Washing with PBST for five times, patting dry the liquid in the hole, adding 100 μ L TMB substrate solution, and developing at 37 deg.C for 10 min; add 50. mu.L of stop solution (10% H)2SO4V/v) terminating the reaction; the absorbance at 450nm was measured with a microplate reader.
Selecting clones which have OD values 3 times larger than those of the negative control holes and are obviously inhibited in the plate 1, recording the numbers of the corresponding holes, transferring the bacterial liquid of the corresponding holes in the mother plate into a sterile centrifuge tube, and adding glycerol for freezing and storing for later use.
Second, experimental results
And (3) measuring 96 randomly picked clones on an elution product measuring plate of the third and fourth screening, and carrying out positive screening on phage clone by using indirect phase ELISA (enzyme linked immunosorbent assay), wherein the result shows that 80 clones are obviously combined with the antigen B-BSA. Further, 80 clones were identified by indirect competitive phase ELISA, and the results showed that 40 positive clones were competitively inhibited from binding to hapten B-BSA by the added fenitrothion. The 40 E.coil TG1 strains are 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. Antibodies with the same sequence are classified into one group, and 10 strains with different gene sequences are obtained in total. The positive clone obtained by screening expresses the nano antibody, and the obtained nano antibody with different amino acid sequences is subjected to working concentration and IC by an ELISA method50A special featureAnd (3) analyzing the specificity, carrying out comparative evaluation on the performance of the antibody, preferably selecting a nano antibody sm6 (the amino acid sequence is shown as SEQ ID NO. 1) from the antibody for detecting fenitrothion, and preparing a fenitrothion detection kit.
Wherein the nano antibody sm6 obtained by sequencing has an amino acid sequence shown in SEQ ID NO. 1. And the nanobody sm6 comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the 4 framework regions and the 3 complementarity determining regions are arranged in the sequence of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4; the amino acid sequence of FR1 is shown as SEQ ID NO.2, the amino acid sequence of FR2 is shown as SEQ ID NO.3, the amino acid sequence of FR3 is shown as SEQ ID NO.4, the amino acid sequence of FR4 is shown as SEQ ID NO.5, the amino acid sequence of CDR1 is shown as SEQ ID NO.6, the amino acid sequence of CDR2 is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 8.
Meanwhile, the nucleotide sequence of the gene for coding the nano antibody sm6 is shown in SEQ ID NO. 9.
Example 3 expression of Nanobodies in the form of Single Domain antibodies
The plasmid of the E.coli TG1 strain of the expression vector carrying the gene of the nano antibody sm6 (the amino acid sequence is shown as SEQ ID NO. 1) subjected to gene sequencing is extracted by a kit, transformed into competent E.coil BL21(DE3) and coated on an LB-Agar-Amp plate for culture to obtain a single colony. A single colony was inoculated into 10mL of LB-Amp, and cultured overnight at 37 ℃ and 250 rpm. The overnight cultures were incubated at 1: 100 in 100mL LB-Amp, 37 degrees, 250rpm culture to logarithmic phase, adding IPTG to the working concentration, 37 degrees, 250rpm culture overnight. Centrifuging the bacterial liquid, removing the supernatant, dispersing the precipitate in 10mL of PBS again, freezing at-80 ℃ for 30min, thawing in 37 ℃ water bath, repeating the process for 3 times, performing ultrasonic treatment for 30min, shaking at 250rpm for 2h to fully extract protein, centrifuging to leave the supernatant, and purifying the supernatant through nickel-agarose gel affinity chromatography to obtain the nano antibody shown in figure 2.
Example 4 Nanobody working concentration determination based on Indirect ELISA method
1. Immobilization of antigens
The fenitrothion B-BSA was diluted to 0.5. mu.g/mL with PBS according to the method of "1, affinity panning of Nanobodies (1) detection of antigen immobilization" in example 2, and the detection of antigen B-BSA was carried out on an ELISA plate.
2. Determination of working concentration of antibody
Example 3 purified nanobody sm6 (amino acid sequence shown in SEQ ID NO. 1) was diluted to 100. mu.g/mL with PBS and then diluted with PBS in a two-fold gradient to give a series of nanobodies of different concentrations (500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.3ng/mL, 15.6ng/mL, 7.8ng/mL, 0 (blank control), 8 concentrations). 50 mu L of PBS is added into each hole in advance, 50 mu L of nano antibody sm6 (amino acid sequence is shown as SEQ ID NO. 1) after gradient dilution is added, 3-hole repeated tests are carried out on the concentration of each nano antibody sm6 (amino acid sequence is shown as SEQ ID NO. 1), and 3 holes are used for blank control (100 mu L of PBS). After incubation at 37 ℃ for 30min, the plates were washed 5 times with PBST, and 100. mu.L of HRP-labeled anti-HA secondary antibody diluted 5000-fold with PBST was added to each well after blotting onto absorbent paper. Incubating at 37 ℃ for 30min, washing the plate for 5 times by PBST, drying on absorbent paper, adding 100 mu L of TMB developing solution into each hole, incubating at 37 ℃ in a dark place for 10min, adding 50 mu L of stop solution into each hole, and reading the OD value of 450nm on an enzyme labeling instrument.
The concentration of sm6 (the amino acid sequence is shown as SEQ ID NO. 1) with OD450 nm between 1 and 1.5 is the working concentration of the nano antibody. Under the experimental conditions, the working concentration of the nanobody sm6 (the amino acid sequence is shown as SEQ ID NO. 1) is 13 ng/mL.
Example 5 fenitrothion assay kit
Composition of kit
The fenitrothion detection kit comprises: an ELISA plate, a nano antibody sm6 (amino acid sequence is shown as SEQ ID NO. 1), detection antigen B-BSA, 0.01M PBS (pH7.4), PBST (0.01M PBS, 0.05% Tween-20), TMB color development liquid, HRP-labeled anti-HA secondary antibody, and 10% H2SO4(v/v) stop solution
Secondly, use of the kit
The using method of the fenitrothion detection kit comprises the following steps:
s1 test antigen B-BSA is diluted to 0.5. mu.g/ml by PBS, 0.1ml is added to each well of an enzyme label plate, the temperature is kept overnight at 4 ℃, after the next day, PBST is used for washing twice, PBS is used for preparing skimmed milk powder (w/v) with 2 percent in situ as stop solution, 150. mu.L is added to each well, the solution is sealed for 1h at 37 ℃, the sealing solution is discarded, and PBST is used for washing twice. Oven drying at 37 deg.C for 30min, and storing at 4 deg.C;
s2, adding a sample to be tested (blank, negative and positive hole control are simultaneously carried out) into the enzyme label plate, adding 50 mu L of the sample into each hole, diluting a nano antibody sm6 (amino acid sequence is shown as SEQ ID NO. 1) to working concentration (13ng/mL) by PBS, adding 50 mu L of antibody diluent into each hole, incubating for 30min at 37 ℃, and washing the plate for 5 times by PBST;
s3 adding 100 mu L of HRP (horse radish peroxidase) -labeled anti-HA secondary antibody diluted 5000 times by PBST (Poly-p-phenylene benzobisoxazole) into each hole after patting dry on absorbent paper, incubating for 30min at 37 ℃, washing the plate for 5 times by PBST, and patting dry the liquid in the hole;
s4 adding 100 μ L of TMB color developing solution into each well, incubating for 10min at 37 deg.C in dark;
s5 Add 50. mu.L 10% H per well2SO4(v/v) stop solution, and reading the OD value of 450nm on a microplate reader.
EXAMPLE 6 plotting of Standard Curve of assay kit
First, experiment method
The fenitrothion standard drug is diluted into standard diluents with different concentrations by PBS, the concentration of the diluted standard drug is 1000ng/mL, 200ng/mL, 40ng/mL, 8ng/mL, 1.6ng/mL, 0.32ng/mL, 0.064ng/mL and 0, and 8 concentrations are added in turn into the enzyme label plate coated by the detection kit in the example 5, the amount of the standard drug is 50 muL in each hole, 3-hole repeated tests are carried out on each concentration, and a 3-hole drug blank group (50 muL PBS) is prepared. Nanobodies were diluted to working concentration (13ng/mL) with PBS and 50. mu.L of antibody dilution was added per well. After the detection kit is used for carrying out a detection experiment, an OD value at 450nm is read by using an enzyme-labeling instrument.
The average value of OD450 values of a drug blank group is recorded as B0, the average value of OD450 values under different fenitrothion standard drug concentrations is recorded as Bx, and the Bx/B0 ratio under different drug concentrations and the standard deviation of parallel data of each group are calculated by using Excel. And drawing a scatter diagram in Origin software and fitting a logistic function by taking the concentration of the fenitrothion medicament as an abscissa and the Bx/B0 ratio as an ordinate.
Second, experimental results
The standard curve is shown in FIG. 3 according to IC50Defining, calculating the corresponding abscissa value when the inhibition rate is 50% on a standard curve by using origin software, wherein the detection sensitivity of the nano antibody sm6 (the amino acid sequence is shown as SEQ ID NO. 1) to fenitrothion is 4.0 ng/mL. According to IC10Defining, calculating the corresponding abscissa value when the inhibition rate is 10% on a standard curve by using origin software, and the detection limit of the nano antibody sm6 on fenitrothion is 0.13 ng/mL.
Example 7 detection kit specificity exploration
First, experiment method
Preparing other 8 fenitrothion and analogue standard solutions thereof, using the detection kit in the example 5, determining other 8 analogues, and reading OD value at 450nm by using a microplate reader. Drawing a standard curve and calculating IC50The value and cross-reactivity are calculated as follows:
second, experimental results
TABLE 4 sensitivity and specificity of the nano antibody sm6 ic-ELISA method for detecting the analog pesticide
The specific determination result is shown in Table 4, the cross reaction rate of the nano antibody sm6 (the amino acid sequence is shown in SEQ ID NO. 1) with fenitrothion is 100%, the cross reaction rate with methyl parathion is 21.4%, the cross reaction rate with parathion is 2.1%, and the cross reaction rate with other 6 pesticide analogs is lower than 1%. The result shows that the nano antibody sm6 can specifically recognize fenitrothion.
Example 8 determination of the Activity of Nanobodies in organic solvents of different concentrations
First, experiment method
The PBS in the detection kit of the embodiment 5 is replaced by mixed liquor (v/v) of acetonitrile with different concentrations (0%, 10%, 20%, 30%, 40% and 50%) and mixed liquor (v/v) of acetone with different concentrations (0%, 10%, 20%, 30%, 40% and 50%) with PBS as a nano antibody sm6 (the amino acid sequence is shown in SEQ ID NO. 1) and a fenitrothion standard diluent. Nanobodies were diluted to working concentration (13 ng/mL). The detection kit in the embodiment 5 is used for respectively measuring the binding capacity of the nano antibody sm6 and the detection antigen B-BSA and the fenitrothion standard drug (20ng/mL), and evaluating the tolerance capacity of the nano antibody to different organic solvents and the same organic solvent at different concentrations.
Second, experimental results
The determination result is shown in FIG. 4, the binding activity of the nano antibody sm6 (the amino acid sequence is shown in SEQ ID NO. 1) and the detection antigen B-BSA in a low-concentration organic solvent (less than 30% of acetone and less than 20% of acetonitrile) is less influenced, but the binding activity with the small molecule analyte is sharply reduced along with the increase of the concentration of the organic solvent. When the proportion of acetone in the organic solvent mixed solution reaches 40 percent and the proportion of acetonitrile reaches 30 percent, the phenomenon that the drug can not inhibit the combination of the antibody and the detection antigen B-BSA, and the signal value does not decrease and reversely rises occurs. The method shows that the nano antibody sm6 (the amino acid sequence is shown as SEQ ID NO. 1) has poor tolerance to two organic solvents commonly used in pretreatment of acetonitrile and acetone in a standard method, the concentration of the organic solvent in an antibody working solution is reduced as much as possible, and the influence of the high-concentration organic solvent on the sensitivity of the antibody is avoided.
Example 9 Activity assay of Nanobodies at different temperatures
First, experiment method
The method comprises the steps of diluting a nano antibody sm6 (amino acid sequence is shown as SEQ ID NO. 1) and a fenitrothion standard substance by PBS, diluting the nano antibody sm6 to a working concentration (13ng/mL), respectively measuring the nano antibody sm6 and a detection antigen B-BSA and a fenitrothion standard drug (20ng/mL) by using the kit of example 5, and detecting the binding activity of the nano antibody sm6 and the detection antigen B-BSA and the fenitrothion standard drug (20ng/mL) by ICELISA (enzyme-linked immunosorbent assay) at different temperatures (4 ℃, room temperature, 37 ℃, 50 ℃, 60 ℃) for a certain time (10min, 20min, 30min, 40min) and the detection antigen B-BSA and the fenitrothion standard drug.
Second, experimental results
The determination result is shown in FIG. 5, the binding activity of the nano antibody sm6 (the amino acid sequence is shown in SEQ ID NO. 1) and the detection antigen is the highest at 37 ℃, but the binding activity with the small molecule analyte is in a descending trend along with the temperature increase. The nano antibody sm6 can work at room temperature in order to improve the sensitivity of detecting the small molecule analyte, and an incubation device is not needed.
Example 10 determination of the Activity of Nanobodies at different pH conditions
First, experiment method
0.01M PBS at different pH values (5.4, 6.4, 7.4, 8.4, 9.4) was used as the antibody and fenitrothion standard diluent instead of PBS in the assay kit of example 5. Nanobodies were diluted to working concentration (13 ng/mL). The detection kit in example 5 is used for respectively measuring the binding capacity of the antibody and the detection antigen B-BSA and the fenitrothion standard drug (20ng/mL), and evaluating the binding activity of the nano antibody and the detection antigen B-BSA and the fenitrothion standard drug in PBS solutions with the same ion concentration and different pH values.
Second, experimental results
The determination result is shown in FIG. 6, and the binding activity of the nano antibody sm6 (the amino acid sequence is shown in SEQ ID NO. 1) and the detection antigen and the micromolecule analyte is slightly reduced along with the increase of pH within the pH range of 5.4-9.4. In general, the nanobody sm6 can maintain better antigen binding activity in a wider pH range, but is more suitable for working in a weakly acidic environment.
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> preparation and application of specific recognition fenitrothion nano antibody
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Gly Val His
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Ala Met Gly Trp Tyr Thr Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Gly Ile Ser Ala Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Lys Phe Gly Leu Arg Ile Phe Gly Ile Gln Glu Arg Pro Asp His
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala His His Ser Glu
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130
<210> 2
<211> 25
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<213> Artificial Sequence (Artificial Sequence)
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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 3
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Gly Trp Tyr Thr Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
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Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
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35
<210> 5
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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala His His Ser Glu
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Ala Ala Lys Phe Gly Leu Arg Ile Phe Gly Ile Gln Glu Arg Pro Asp
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caggtgcagc tcgtggagtc tgggggaggc ttggtgcagg ctgggggctc tctgagactg 60
tcctgtgcag cctctggact caccttcggt gtacatgcca tgggctggta cacccaggct 120
ccagggaagg agcgcgaatt ggtcgcaggt attagtgctg gtggtaccac atactatgca 180
gactctgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
cagatgaaca gcctgaaacc tgaggacacg gccgtctatt actgtgcagc caaattcggt 300
ctacgaattt tcggtatcca agaaaggccg gaccactggg gccaggggac ccaggtcacc 360
gtctcctcag cgcaccacag cgaagacccc cat 393
Claims (10)
1. A nano antibody is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1.
2. A gene for coding a nano antibody is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 9.
3. A recombinant vector comprising the gene of claim 2 linked thereto.
4. A recombinant cell comprising the expression vector of claim 3 or capable of expressing the nanobody of claim 1.
5. The use of one or more of the nanobody of claim 1, the gene of claim 2, the recombinant vector of claim 3 or the recombinant cell of claim 4 in immunological detection analysis for detecting fenitrothion.
6. A method for detecting fenitrothion for non-diagnostic purposes, which is characterized in that the nano-antibody of claim 1 is used.
7. The detection method according to claim 6, characterized in that the detection is carried out based on an indirect ELISA method using the fenitrothion complete antigen of formula (I) as a detection antigen,
8. a kit for detecting fenitrothion, which is characterized by comprising the nano antibody of claim 1.
9. The kit of claim 8, further comprising the fenitrothion complete antigen of formula (I),
10. the kit of claim 8, further comprising an enzyme-labeled antibody, a color-developing agent, and a stop solution.
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