CN110577601A

CN110577601A - Chlorothalonil anti-idiotype single domain antibody library, anti-idiotype single domain antibody and application thereof

Info

Publication number: CN110577601A
Application number: CN201910838114.6A
Authority: CN
Inventors: 赵岩岩; 赵圣明; 王宝石; 谭凤玲; 李红波; 周威; 娄文娟
Original assignee: Henan Institute of Science and Technology
Current assignee: Henan Institute of Science and Technology
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2019-12-17

Abstract

The invention discloses a chlorothalonil anti-idiotype single domain antibody library, an anti-idiotype single domain antibody and application thereof, belonging to the technical field of genetic engineering and immune engineering. The invention obtains chlorothalonil single-domain antibodies by screening a chlorothalonil single-domain antibody library, immunizes mice again, takes spleens of the mice to extract RNA, and constructs the chlorothalonil anti-idiotype single-domain antibody library by using Escherichia coli TG1 as host bacteria and a phage display technology. The invention utilizes the phage screening technology to screen the antibody library to obtain the anti-idiotype single domain antibody which has competitive binding activity with chlorothalonil, saves the cost of antibody preparation, is easy to prepare a large amount of antibodies and is convenient for the later modification of the obtained antibodies; provides a green immunodetection method for the detection of chlorothalonil, reduces the harm of the detection method to operators, reduces the use of organic substances and has very wide application prospect.

Description

chlorothalonil anti-idiotype single domain antibody library, anti-idiotype single domain antibody and application thereof

Technical Field

the invention relates to the technical field of genetic engineering and immune engineering, in particular to a chlorothalonil anti-idiotype single domain antibody library, an anti-idiotype single domain antibody and application thereof.

background

Chlorothalonil is a substituted benzene bactericide, has the chemical name of 2,4,5, 6-tetrachloro-1, 3-phthalonitrile and the molecular formula of C₈Cl₄N₂. The action mechanism of chlorothalonil is mainly to destroy the activity of glyceraldehyde triphosphate dehydrogenase of fungal cells, so that the normal metabolism of the fungal cells is destroyed, and the chlorothalonil is widely applied to the control of fungal diseases on vegetables, fruits, grains and economic crops. Chlorothalonil has certain acute and chronic toxicity, is relatively stable in the environment and has obvious accumulative toxicity. The problem of the residue of chlorothalonil has also attracted a great deal of attention due to its widespread use.

The detection of the pesticide micromolecules can adopt an immunoassay method in addition to the detection of a precise instrument, the detection sensitivity and accuracy of the precise instrument are high, but the pretreatment of samples is complex, the requirement on equipment is high, the detection cost is high, and the advantages are not obvious when a large number of samples are detected; the immunoassay method can exactly make up for the defects of the detection of a precise instrument and realize the rapid, economical and high-flux detection of the sample. Antibodies are important components of immunoassays, and complete antibodies are composed of two heavy chains and two light chains, and the variable regions of the heavy and light chains play a key role in antigen binding. In addition to intact antibodies, fragments of antibodies such as Fab, scFv, DAB can also be used for immunoassays.

There are three types of antibody determinants: isotype, allotype, and idiotype. The isotype determines the antibody type and subtype; allotypes determine the diversity of antibodies between species; idiotypes determine the binding specificity to different antigens. Oudin et al discovered an epitope in 1963 that was different from either the isoform or the allotype, and called idiotype, meaning that the individual genotype, now translated to the idiotype. In 1974, Jerne, Denmark, proposed the concept of immune network that the body was stimulated by foreign antigens, lymphocytes were activated to produce antibody Ab1, and lymphocytes recognizing Ab1 were activated to produce anti-idiotypic antibody Ab2, and antibodies Ab3 and Ab4, etc., which form a complex Id and anti-Id network in vivo. Ab2, which recognizes the idiotypic determinant of Ab1, is an anti-idiotypic antibody to the antigen. The anti-idiotype antibody can be used as a substitute of an antigen or a standard substance in an immunoassay method, a green immunoassay method is established, and a new way is provided for antigen synthesis. There are three types of anti-idiotype antibodies: polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies. The phage display technology is a gene antibody technology which is mature and has relatively wide application at present. In the 80 s of the 20 th century, a phage display technology is gradually established and developed, the system can express antibody genes on the surface of recombinant phage capsid protein, and a target antibody is obtained through separation by multiple rounds of affinity screening.

The advantage of the phage display technology is mainly embodied in the unification of genotype and phenotype, the phage display technology takes phage or phagemid as a carrier, exogenous polypeptide or protein genes are integrated into a phage genome, and the exogenous genes are expressed in a fusion form and displayed on the surface of the phage, so that the synchronous screening of protein (phenotype) and genotype is realized. However, the construction research of the chlorothalonil anti-idiotype antibody library is blank at present; therefore, it is a problem to be solved by the skilled person to provide a chlorothalonil anti-idiotype single domain antibody library and anti-idiotype single domain antibodies and uses thereof.

Disclosure of Invention

In view of the above, the present invention provides a chlorothalonil anti-idiotype single domain antibody library, an anti-idiotype single domain antibody and applications thereof, provides a chlorothalonil anti-idiotype single domain antibody library for immunodetection of chlorothalonil, and establishes a chlorothalonil green immunodetection method using screened anti-idiotype single domain antibodies.

In order to achieve the purpose, the invention adopts the following technical scheme:

The chlorothalonil single domain antibody of the invention is chlorothalonil-NH₂KLH is used as immunogen to immunize mice, a murine heavy chain antibody library is constructed, and chlorothalonil-OH-OVA is used as a coating antigen to be screened and obtained.

Screening of anti-idiotype antibodies: amplifying by adopting a method combining negative screening and competitive elution to obtain an anti-idiotype single domain antibody library, coating a 6-hole plate with a chlorothalonil single domain antibody to be used as a positive hole, using an uncoated hole as a negative hole, sealing for 2h by using 2% MPBS (multi-path polymerase chain reaction), firstly adding the amplified anti-idiotype antibody library into the negative hole, incubating for 1h, carefully sucking out the antibody to be added into the positive hole to incubate for 1h, washing the unbound phage, competitively eluting the phage bound with the chlorothalonil single domain antibody by using the chlorothalonil single domain antibody, and obtaining the high-affinity anti-idiotype antibody through 3 rounds of screening.

Soluble expression of anti-idiotype antibodies: extracting positive clone plasmid, connecting to pET26b plasmid after enzyme cutting, selecting colibacillus BL21 as host bacterium, carrying out induction expression by IPTG, and purifying expression periplasm by nickel column to obtain soluble anti-idiotype antibody.

Use of anti-idiotype single domain antibodies: establishing a chlorothalonil competitive green immunodetection method, performing four-parameter fitting by utilizing origin software to obtain a competitive inhibition curve, establishing a regression equation, and calculating IC₅₀。

According to the technical scheme, compared with the prior art, the invention discloses and provides a chlorothalonil anti-idiotype single domain antibody library, an anti-idiotype single domain antibody and application thereof, after the chlorothalonil single domain antibody is obtained by screening the chlorothalonil single domain antibody library, a mouse is immunized again, the spleen of the mouse is taken to extract RNA, and the phage display technology is utilized, escherichia coli TG1 is taken as host bacteria, so that the chlorothalonil anti-idiotype single domain antibody library is constructed. The invention utilizes the phage screening technology to screen the antibody library to obtain the anti-idiotype single domain antibody which has competitive binding activity with chlorothalonil, saves the cost of antibody preparation, is easy to prepare a large amount of antibodies and is convenient for the later modification of the obtained antibodies; provides a green immunodetection method for the detection of chlorothalonil, reduces the harm of the detection method to operators, reduces the use of organic substances and has very wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of the synthesis route of chlorothalonil immunogens according to the present invention;

FIG. 2 is a graph of the ultraviolet spectrum of chlorothalonil immunogens of the present invention;

FIG. 3 is a schematic diagram of the synthetic route of chlorothalonil envelope of the present invention;

FIG. 4 is a diagram of an original UV spectrum of chlorothalonil coating according to the present invention;

FIG. 5 is a diagram showing the result of total RNA electrophoresis detection in the preparation of a chlorothalonil single domain antibody according to the present invention;

FIG. 6 is an electrophoretogram of the heavy chain variable region gene of the present invention;

Wherein 1-8 are 8 repeats; CK is that distilled water is adopted to replace a template;

FIG. 7 is a graph showing the screening results of partial monoclonal ELISA using chlorothalonil single domain antibodies of the present invention;

Wherein, 1-4 are different selected monoclonals, dark grey is a positive pore value, and light grey is a negative pore value;

FIG. 8 is the PCR identification result of the chlorothalonil single domain antibody soluble expression recombinant bacteria of the invention;

Wherein, 1-4 are four selected monoclonals; CK is that distilled water is adopted to replace a template;

FIG. 9 is an electrophoretogram of purified protein of a chlorothalonil single domain antibody of the invention;

FIG. 10 is a diagram showing the result of the total RNA electrophoresis detection of the anti-idiotype antibody according to the present invention;

wherein 1-3 are 3 repeats;

FIG. 11 is a diagram showing the PCR results of the anti-idiotype single domain antibody heavy chain variable region gene of the present invention;

Wherein, 1-7 is a target band obtained by amplification; CK is distilled water to replace a template;

FIG. 12 is a diagram showing the screening of a part of cloning ELISA results for an anti-idiotype single domain antibody according to the present invention;

FIG. 13 shows the PCR identification result of the anti-idiotype single domain antibody soluble expression recombinant bacteria of the present invention;

Wherein, 1-5 are four selected monoclonals; CK is that distilled water is adopted to replace a template;

FIG. 14 is an electrophoretogram of purified protein of an anti-idiotype single domain antibody of chlorothalonil in accordance with the present invention;

FIG. 15 is a graph showing competitive inhibition curves according to the present invention.

Detailed Description

the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

(I) obtaining of chlorothalonil Single Domain antibodies

EXAMPLE 1 Synthesis of immunogen and coatingen

according to a hapten design principle, structural modification is carried out on chlorothalonil, amino functional groups and hydroxyl functional groups are respectively introduced firstly, and then KLH (Keyhole Lippethylocyanin) and OVA (Ovalbumin) are respectively introduced to serve as complete antigens, wherein a specific synthetic route is as follows:

Synthetic route of immunogen: weighing 200mg of chlorothalonil, dissolving in 10mL of dichloromethane, adding 10mL of ethylenediamine and 40mL of water, stirring at room temperature for 24 hours,washing the dichloromethane layer with water, and then distilling under reduced pressure to remove the solvent to obtain a yellow-green residue which is CTN-NH₂. Synthesis of CTN-NH by glutaraldehyde method₂KLH (immunogen), scheme see FIG. 1. 50mg of CTN-NH are weighed₂Dissolving in 4mL DMF, adding 5mL PBS solution of 9mg/mL KLH, stirring, adding 40 μ L glutaraldehyde dropwise, stirring at 4 deg.C overnight, centrifuging to remove precipitate, dialyzing, and lyophilizing. The UV scan is shown in FIG. 2.

Synthetic route of coating source: weighing 200mg of chlorothalonil and 76mg of potassium fluoride, dissolving in 8mL of glycol solution, heating at 90 ℃ for 24h, cooling, adding 10g of ice water, separating precipitate, and air-drying to obtain a white solid, namely CTN-OH. The CTN-OH-OVA (coating antigen) is synthesized by carbonyl imidazole method, and the reaction route is shown in figure 3. Dissolving 50mg of CTN-OH in 2mL of DMF, adding equal amount of 1, 1' -carbonylimidazole, stirring at room temperature for 3h, slowly adding the reaction solution into OVA carbonate buffer solution with pH of 9.51, magnetically stirring at 4 ℃ for 4h, dialyzing, and freeze-drying for later use. The UV scan is shown in FIG. 4.

Example 2 extraction of RNA and cDNA Synthesis from splenocytes from immunized mice

breeding 6-8 week old Balb/c female white mouse for 1-2 weeks, collecting blood from tail vein 1 week before primary immunization to obtain negative serum, and collecting the negative serum with prepared immunogen (CTN-NH)₂KLH) of mice. First, antigen (CTN-NH)₂KLH) and an equal volume of freund's adjuvant were emulsified by pushing back and forth with a syringe until the mixture was uniformly milky white. When preparing the serum, standing the collected fresh blood at room temperature for 1-2h, standing overnight at 4 ℃, centrifuging at 4 ℃ and 10000g/min for 10min, and collecting the supernatant, namely the multi-antiserum. Measuring the titer of polyclonal antiserum, selecting high-titer mouse, taking out intact spleen, removing fat and connective tissue, cutting, placing into a mortar treated with DEPC water, adding liquid nitrogen, grinding, adding Trizol, standing at room temperature for 5min, adding chloroform into supernatant, mixing, standing for 15min, centrifuging at 12000g for 15min, adding isopropanol into upper water phase, mixing, standing for 10min, suspending the precipitate with 75% ethanol, centrifuging at 8000g for 5min, drying at room temperature, and drying with RNase-free ddH₂Dissolving the precipitate with O water to obtain RNA, and detecting by agarose gel electrophoresis, wherein the electrophoresis result is shown in figure 5. Using reverse transcriptionthe kit (Beijing Solebao science and technology Co., Ltd.) was used to obtain cDNA by reverse transcription according to the instructions.

Example 3 construction of chlorothalonil single domain antibody library

using the cDNA obtained in example 2 as a template, a murine heavy chain variable region gene was amplified using a heavy chain variable region universal primer (see Table 1, wherein HB is an upstream primer and HF is a downstream primer). And (3) PCR reaction system: 2 XMixs (Nanjing Novozam Biotech Co., Ltd.) 25. mu.L, 2.5. mu.L of each of the upstream and downstream primers, and 5. mu.L of the template. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and reaction circulation for 35 times; extension at 72 ℃ for 10 min. The PCR electrophoresis results are shown in FIG. 6. After purifying the PCR product with a PCR purification kit (Beijing Quanji Biotechnology Ltd.), double digestion was performed with Nco I and Not I, the digested PCR product and pR2 vector were ligated with a DNA ligation kit from TaKaRa, and the product was transformed into E.coli TG1 (stored in the laboratory).

TABLE 1 Universal primers for heavy chain variable regions

Note: degenerate base symbols: k is G/T; r is A/G; m is A/C; G/C; y is C/T; w is A/T.

example 4 screening of chlorothalonil single domain antibody libraries

The chlorothalonil single domain antibody library constructed in example 3 was put into 2 XTY-A-G (1L of a medium containing 16G of tryptone, 10G of yeast extract, 5G of NaCl, 100. mu.g/mL of Amp and 50. mu.g/mL of Kan, 1% of glucose), and cultured with shaking at 37 ℃ until OD is reached₆₀₀when nm is 0.4, KM13 helper phage (stored in the laboratory) was added, water bath was carried out at 37 ℃ for 30min, 3200G was centrifuged for 10min, the supernatant was discarded, 500mL of 2 XTY-A-K-G was resuspended, and the mixture was cultured overnight at 25 ℃. Centrifuging the overnight culture 3200g for 30min, taking the supernatant, adding PEG/NaCl, mixing uniformly, and performing ice bath for 1 h; centrifuging at 3200g for 30min, and discarding the supernatant; resuspending the precipitate with 5mL PBS, adding 1mL PEG/NaCl, and ice-cooling for 30 min; centrifuging at 3200g for 30min, and discarding the supernatant; resuspend the pellet in 1mL PBS, centrifuge at 3200g for 5min, and use 0.45Mu.m sterilized filter membrane filtration to obtain phage library. Storing at 4 deg.C for a short time, adding glycerol, and storing in refrigerator at-70 deg.C for a long time.

Coating a 6-pore plate with 1mL of CTN-OH-OVA, and coating overnight at 4 ℃; meanwhile, coating OVA is used as a negative hole for negative screening; washing the wells with PBS buffer for 3 times, and blocking with 2% MPBS overnight; after sealing, washing the wells with PBS for 3 times, adding 1mL of phage library into the negative wells, and incubating for 1h at room temperature under mild oscillation; adding the phage library into the positive hole, adding 2% MPBS at the same time, and incubating for 1h at room temperature under mild oscillation; washing the wells with PBST wash solution 10 times, washing away unbound phage, competitively eluting bound phage with chlorothalonil, and incubating at room temperature for 20 min. Culture of E.coli TG1 to OD_600nm0.4; adding 500 mu L of competitive eluent (PBS solution containing 50 mu g/mL chlorothalonil) into 1.5mL of bacterial liquid, and infecting for 1h in water bath at 37 ℃; after infection, centrifuging 3200G for 5min, resuspending the thallus with 200 μ L of 2 × TY (tryptone 16G, yeast extract 10G and NaCl 5G, constant volume to 1L, autoclaving for use), coating the thallus on TYE-A-G (autoclaved TYE medium, cooling to about 60 deg.C, adding Amp and 1% glucose at final concentration of 100 μ G/mL, mixing, and then pouring onto a plate, wherein TYE is tryptone 10G, yeast extract 5G, NaCl 8G, agar powder 15G, constant volume to 1L, autoclaving for use), culturing overnight at 37 deg.C. The titer of the eluate was also determined. The overnight grown cells were scraped off the TYE-A-G plate with 2mL of 2 XTY, and half of the cells were added to 500mL of 2 XTY-A-G (4%) and cultured to OD_600nmAdding KM13 helper phage for infection, and culturing overnight after infection; the other half was stored at-70 deg.C; supernatants from overnight cultures were precipitated with PEG/NaCl and the amplified pools were used for the next round of screening, for a total of four rounds of screening, with the input-output ratio for the four rounds of screening shown in Table 2.

TABLE 2 output-to-input ratio for four-round screening

Example 5 verification of Positive clones

The single clones obtained in the last round of screening of example 4 were picked and analyzed for ELISA bindingAnd (5) fruit. Taking the hole coated with CTN-OH-OVA as a positive hole and the hole coated with OVA as a negative hole, washing the plate for 3 times by PBST the next day, and sealing for 2h at 37 ℃ by using OVA; washing the plate, adding phage, and incubating for 1 h; washing the plate, adding an HRP-labeled anti-M13 enzyme-labeled secondary antibody, and incubating for 1 h; washing the plate, adding TMB color development liquid, developing for about 15min, and adding 2mol/L sulfuric acid to terminate the reaction; OD determination by means of enzyme-linked immunosorbent assay_450nm. OD-positive/OD-negative>Clones of 2.1 were considered positive clones. Partial ELISA results are shown in FIG. 7, where 1-4 are different singles picked, dark grey is the positive well value and light grey is the negative well value. The monoclonal identified as positive by ELISA was subjected to PCR verification, while E.Coli TG1 was used as a negative control, the primers are shown in Table 3, the reaction system is shown in Table 4, and the PCR conditions were: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 40s, and extension at 72 ℃ for 40s, repeating for 30 cycles; extension was carried out at 72 ℃ for 10 min. As a result, a band was found to be evident at about 350bp, confirming that the clones obtained by screening exhibited complete single-domain antibody fragments.

table 3 verification of primer sequences

TABLE 4PCR reaction System

Example 6 soluble expression of chlorothalonil single domain antibodies

The positive clones obtained in example 5 were cultured overnight, plasmids of overnight bacteria were extracted using a small plasmid extraction kit, and then PCR amplification was performed (primers and system are shown in tables 3 and 4, and reaction conditions are shown in example 5) to obtain a large number of target bands. And purifying the PCR product by using a PCR product purification kit. The PCR-purified product and the pET26b plasmid were double digested (Nco I and Not I), digested overnight at 37 ℃ and incubated at 80 ℃ for 1 hour to inactivate the enzyme. And (3) carrying out glue recovery on the large fragment of the enzyme digestion plasmid by using a glue recovery kit, and purifying the enzyme digestion product of the PCR product by using a PCR product purification kit. The digested plasmid and the purified target fragment after gel recovery are connected overnight at 16 ℃ by T4DNA ligase. 100 mu L of the ligation product was transformed into 100 mu L of L.coli BL21 competent cells, incubated on ice for 30min, heat-shocked at 42 ℃ for 60s, ice-bathed again for 2min, 900 mu L of 2 XTY was added, resuscitated at 37 ℃ for 1h at 250rpm, an appropriate amount of culture medium was taken and coated with a plate of TYE-K (TYE-K: autoclaved TYE medium cooled to about 60 ℃ and mixed with Kna at a final concentration of 50. mu.g/mL, and the plate was poured over overnight). Recombinant bacteria were picked from overnight-cultured plates, and subjected to PCR identification after liquid culture (primers and system are shown in tables 3 and 4). The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, and 30 cycles in total; and finally, extending for 10min at 72 ℃. The results of electrophoresis are shown in FIG. 8, where 1-4 are four single clones picked. And (3) verifying that the strain containing the complete target fragment is cultured overnight through PCR, extracting plasmids, and then performing double enzyme digestion identification and PCR identification. Carrying out IPTG induced expression on the recombinant bacteria, culturing the recombinant bacteria overnight, taking 500 microliter to transfer into 50mL of 2 XTY-K-G liquid culture medium, culturing at 37 ℃ and 250rpm until the late logarithmic phase, adding IPTG with the final concentration of 1mmol/L, and inducing expression at 30 ℃ and 200rpm overnight. Centrifuging at 10000g for 10min to collect overnight cultured thallus, adding 5mL TES, ice-cooling for 30min, adding 12mL sterile water, ice-cooling for 30min, centrifuging at 10000g for 10min, and collecting supernatant containing single domain antibody. The monoclonal antibody was purified by a nickel affinity column to obtain a chlorothalonil single domain antibody, and the electrophoretogram of the purified protein is shown in FIG. 9.

(II) construction of a library of anti-idiotypic antibodies of chlorothalonil and obtaining of anti-idiotypic antibodies of chlorothalonil

example 7 extraction of RNA and cDNA Synthesis from splenocytes from immunized mice

A chlorothalonil single-domain antibody is used for immunizing a mouse, and an antigen and an adjuvant are emulsified to obtain a water-in-oil emulsion. Collecting blood after one week of immunization, measuring titer of polyclonal antiserum, selecting high titer mouse, collecting spleen, removing fat and connective tissue, cutting spleen into pieces, grinding in a mortar containing liquid nitrogen, adding Trizol, standing at room temperature for 5min, collecting supernatant, adding chloroform, mixing, standing for 15min, centrifuging at 12000g for 15min, adding isopropanol to the water, collecting supernatant, and collecting supernatantMixing, standing for 10min, resuspending the precipitate with 75% ethanol, centrifuging at 8000g for 5min, drying at room temperature, and precipitating with RNase-free ddH₂Dissolving in O water to obtain a solution containing RNA, and detecting by agarose gel electrophoresis, wherein the result is shown in FIG. 10. And (3) carrying out reverse transcription by using a reverse transcription kit according to the reverse transcription kit instruction, and obtaining cDNA by reverse transcription.

EXAMPLE 8 construction of anti-idiotype antibody libraries

using the cDNA synthesized in example 7 as a template, a heavy chain variable region universal primer was used to amplify to obtain a heavy chain variable region gene, and the PCR reaction conditions for heavy chain variable region were: firstly, pre-denaturation is carried out at 95 ℃ for 5 min; ② denaturation at 95 ℃ for 30 s; ③ 57 ℃ annealing for 1 min; extension at 72 ℃ for 1 min; reaction is circulated for 30 times; extension for 10min at 72 deg.C. The PCR amplification results are shown in FIG. 11.

purifying the PCR product by using a PCR purification kit, carrying out double enzyme digestion on the purified PCR product by using Nco I and Not I, connecting the PCR product subjected to enzyme digestion and a pR2 vector by using a DNA ligation kit of TaKaRa company, and electrically transforming the constructed vector into escherichia coli TG1 by using an electrotransformation machine to construct an anti-idiotype antibody library.

Example 9 screening of anti-idiotype Single Domain antibodies

The anti-idiotype single domain antibody library obtained in example 8 was inoculated into 2 XTY-A-G medium and cultured with shaking at 37 ℃ until OD_600nmWhen the amount of the phage was 0.4, the helper phage KM13 was added, the mixture was washed with water at 37 ℃ for 30min, and 3200G was centrifuged for 10min, and the supernatant was discarded, and the pellet was resuspended in 500mL of 2 XTY-A-K-G and incubated at 25 ℃ overnight. Centrifuging the overnight culture 3200g for 30min, taking the supernatant, adding one-fourth volume of PEG/NaCl, mixing uniformly, and performing ice bath for 1 h; centrifuging again, discarding the supernatant, resuspending the pellet in 5mL PBS, adding 1mL PEG/NaCl, resuspending the pellet in 1mL PBS, centrifuging at 3200g for 5min to remove residual cell debris, and filtering with 0.45 μm sterile filter. Storing at 4 deg.C for a short time, adding glycerol, and storing in refrigerator at-70 deg.C for a long time.

Coating a 6-well plate with 1mL of chlorothalonil single-domain antibody, and coating overnight at 4 ℃; coating skim milk powder as negative holes to carry out negative screening; washing wells with PBS buffer for 3 times, and blocking with 2% MPBS overnight; after blocking, wash the wells 3 times with PBSAdding 1mL of phage library into the negative hole, and incubating for 1h at room temperature in a shaking way; sucking out the phage library from the negative hole, adding the phage library into the positive hole, adding 2% MPBS, and performing oscillation incubation for 1h at room temperature; washing the wells with PBST washing solution 15 times, washing away the unbound phage, performing competitive elution of the bound phage with chlorothalonil single domain antibody, and incubating for 20min at room temperature. Coli TG1 to OD were cultured while the screening was performed_600nm0.4; adding 500 mu L of competitive eluent into 1.5mL of fresh bacterial liquid, and infecting for 1h in water bath at 37 ℃; 3200G was centrifuged for 5min to collect the cells, 200. mu.L of 2 XTY was resuspended, and the suspension was spread on TYE-A-G plates and incubated overnight at 37 ℃. Simultaneously, the eluent titer determination is carried out. The overnight cultured cells were scraped off the TYE-A-G plate with 2mL of 2 XTY medium, and half of the scraped cells were added to 500mL of 2 XTY-A-G medium and cultured to OD_600nmAdding a helper phage KM13 after being 0.4, transferring the infected phage KM13 into a 2 XTY-A-K-G culture medium for overnight culture; the other half was stored at-70 deg.C; supernatants from overnight cultures were precipitated with PEG/NaCl and the amplified pools were used for the next round of screening, for a total of four rounds of screening. The four screening input-output ratios are shown in table 5.

TABLE 5 output-to-input ratio for four rounds of screening

EXAMPLE 10 verification of Positive clones

Example 9 clones obtained from the last round of screening were subjected to ELISA validation and analyzed for ELISA results. Taking the hole coated with the chlorothalonil single domain antibody as a positive hole and the hole coated with the skimmed milk powder as a negative hole, and after PBST washing the plate for 3 times the next day, sealing the plate for 2 hours at 37 ℃ by using 2% skimmed milk powder; washing the plate with PBST, adding amplified phage, and incubating for 1 h; washing the PBST plate, adding an HRP-labeled anti-M13 enzyme-labeled secondary antibody, and incubating for 1 h; PBST washing plate, adding TMB developing solution, developing for about 15min, adding 2M sulfuric acid to terminate reaction; OD determination by means of enzyme-linked immunosorbent assay_450nm. OD-positive/OD-negative>2.1 is a positive clone. The ELISA assay results are shown in FIG. 12. Performing PCR verification on the monoclonal identified as positive by ELISA, and simultaneously using E.coli TG1 as a negative control to verify whether the clones obtained by screening are positive or notIntact antibody fragments are shown. As a result, a band was found to be evident at about 350bp, confirming that the clones obtained by the screening exhibited the entire anti-idiotype single domain antibody fragment.

Example 11 soluble expression of anti-idiotype Single Domain antibodies

The positive clones obtained in example 10 were cultured overnight, plasmids were extracted using a plasmid miniprep kit, and PCR amplification was performed to obtain a large number of target bands. It was purified using a PCR product purification kit. The PCR purified product and the pET26b plasmid were subjected to double digestion, digested at 37 ℃ overnight, and inactivated at 80 ℃ for 1 hour. And (3) carrying out glue recovery on the large fragment of the enzyme digestion plasmid by using a glue recovery kit, and purifying the enzyme digestion product of the PCR product by using a PCR product purification kit again. The digested plasmid and the purified target fragment after gel recovery are connected by T4 ligase and are connected overnight at 16 ℃. Taking the ligation product transformed E.coliBL21 competent cells, resuscitating at 37 ℃ and 250rpm for 1h, sucking a proper amount of culture solution to coat the TYE-K plate, and culturing overnight. Recombinant bacteria are selected, liquid culture is carried out, bacteria liquid PCR identification is carried out, and identification results are shown in figure 13. The strain containing the complete target fragment is cultured overnight through PCR verification, and double enzyme digestion identification is carried out after plasmid extraction. And (3) carrying out IPTG induced expression on the verified recombinant bacteria, culturing the recombinant bacteria overnight, taking 1mL of the cultured recombinant bacteria, transferring the cultured recombinant bacteria into 100mL of 2 XTY-K-G liquid culture medium, culturing the cultured recombinant bacteria at 37 ℃ and 250rpm until the late logarithmic phase, and adding IPTG with the final concentration of 0.6mM and carrying out induced expression at 30 ℃ and 200rpm for 18 h. Centrifuging at 10000g for 10min to collect thallus, adding 10mL TES, ice-cooling for 30min, adding 24mL sterile water, ice-cooling for 30min, centrifuging at 10000g for 10min, and collecting supernatant as periplasm of the anti-idiotype single domain antibody. Purifying with His-trap FF purification column to obtain chlorothalonil anti-idiotype single domain antibody, and obtaining purified protein electrophoresis chart shown in figure 14.

EXAMPLE 12 use of anti-idiotype Single Domain antibodies

mu.L of chlorothalonil single domain antibody was coated on ELISA plates overnight at 4 ℃ and blocked for 1h at 37 ℃ with 2% MPBS. 50 μ L of a serially diluted multiple (0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 μ g/mL) chlorothalonil standard was mixed well with 50 μ L of the anti-idiotype single domain antibody, added together to the ELISA plate and incubated for 1h at room temperature.washing the plate with PBST, adding 100 μ L of 1:5000 HRP-labeled anti-His enzyme-labeled secondary antibody, incubating at room temperature for 1h, washing the plate again, adding 100 μ L of TMB developing solution, developing for 15min, adding 50 μ L of 2mol/L sulfuric acid to terminate the reaction, and reading OD with enzyme-labeling instrument_450nm. Performing four-parameter fitting by using origin software to obtain a competitive inhibition curve, establishing a regression equation, and calculating IC₅₀. The fitted curve is shown in FIG. 15, IC₅₀It was 0.262. mu.g/mL.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> institute of science and technology of Henan

<120> chlorothalonil anti-idiotype single domain antibody library, anti-idiotype single domain antibody and use thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 1

catgccatgg ggctcgtgca ggtkcaggtg magcagtcwc g 41

<210> 2

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 2

catgccatgg ggctcgagct ggarcaggtg rwgsagtcwg g 41

<210> 3

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 3

catgccatgg ggcccgagta ggtcaaactg cagsggyctg g 41

<210> 4

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 4

catgccatgg ggctctagga ggtgmcgctg gtgaartcyg g 41

<210> 5

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 5

catgccatgg ggctcgagga rgtgawgstg gtggagtctg g 41

<210> 6

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 6

catgccatgg ggttcgagga rgtgcagctk cagtagtcag g 41

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 7

catgccatgg ggctcaagga gttccaggtg carcagtcmg krsc 44

<210> 8

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 8

catgccatgg tgcgcaagga ggttcagctk cagcagtctg krgc 44

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 9

catgccatgg gacgccagga rgtkaarctk stmgagtctg g 41

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 10

ttgcggccgc gaattagtkr rsggactgtg agagtggtkc 40

<210> 11

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 11

ttgcggccgc ggtcttgttg mrgaracagt gaccrkrgtc c 41

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 12

ttgcggccgc gcacttgttg tggagacrgt gactgwggtt c 41

<210> 13

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 13

ttgcggccgc gtactagawc magaracrgt rasrgtggtr c 41

<210> 14

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 14

ttgcggccgc gcacaagtyg aggagacggt gaccrkggts c 41

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 15

ctttagtggt acctttctat gcgg 24

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 16

cgaattcaga tcctcttctg aga 23

Claims

1. A chlorothalonil anti-idiotype single domain antibody library is characterized in that a chlorothalonil single domain antibody is taken as an immunogen, the titer is measured after a mouse is immunized, a mouse with high titer is selected, spleen RNA of the mouse is extracted, and the spleen RNA is subjected to reverse transcription to form cDNA; and amplifying the VH gene segment by using a heavy chain universal primer, connecting the VH gene segment with a vector, and transforming the VH gene segment into escherichia coli TG1 to obtain a chlorothalonil anti-idiotype single-domain antibody library.

2. A chlorothalonil anti-idiotype single domain antibody, which is characterized in that a phage library is constructed according to the chlorothalonil anti-idiotype single domain antibody library obtained in claim 1, the chlorothalonil single domain antibody is used as a coating antigen, the single domain antibody is obtained through negative screening and competitive elution combination, and the single domain antibody is subjected to soluble expression to obtain the chlorothalonil single domain antibody.

3. Use of a chlorothalonil anti-idiotype single domain antibody according to claim 2 for the detection of chlorothalonil.