CN113201530A

CN113201530A - Method for removing phage expressing carrier protein nano antibody in hapten phage display nano antibody library

Info

Publication number: CN113201530A
Application number: CN202110492552.9A
Authority: CN
Inventors: 王保民; 何青青; 王伟轩; 赵亚杰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-05-06
Filing date: 2021-05-06
Publication date: 2021-08-03

Abstract

The invention discloses a method for removing phage expressing carrier protein nano-antibody in a hapten phage display nano-antibody library. The method comprises (1) incubating the magnetic beads coupled with the carrier protein and the hapten phage display nano-antibody library to obtain magnetic beads combined with phage of the expression carrier protein nano-antibody; (2) and removing the magnetic beads of the phage combined with the expression carrier protein nano antibody by adopting magnetic separation, thereby removing the phage expressing the carrier protein nano antibody in the hapten phage display nano antibody library. The method improves the screening efficiency of the small molecule nano antibody.

Description

Method for removing phage expressing carrier protein nano antibody in hapten phage display nano antibody library

Technical Field

The invention relates to the technical field of nano antibody preparation, in particular to a method for removing phage expressing carrier protein nano antibody in a hapten phage display nano antibody library.

Background

In 1993, Hamers-Casterman et al found that a specific antibody, named heavy-chain antibodies (HCabs), was present in camelids. This antibody naturally lacks the light chain, the conserved region Fc fragment is identical to a conventional antibody, and the heavy chain lacks the heavy chain constant region CH1, but may exhibit full antibody binding capacity. The antigen-binding variable region of HCAbs can be easily cloned from genes, and this part is a heavy chain single domain antibody (VHH), which is the smallest antibody-binding monomer currently found, has a size of 15kDa, is only 1/10 of conventional antibodies, has a flat and long structure with a diameter of about 25nm and a length of 4nm, and is also called nanobody (Nbs) by the company Ablynx, belgium.

The existing method for preparing the nano antibody is to display the antibody on the surface of filamentous phage by using a phage display technology and then carry out in vitro screening. The small molecule substance has no immunogenicity but only reactogenicity, so the small molecule substance cannot be directly used for immunizing animals to prepare antibodies when preparing the antibodies. It is necessary to prepare complete antigens by coupling small molecules with carrier proteins and then to immunize camelids or sharks with the complete antigens after emulsification with Freund's adjuvant. Then extracting total RNA of alpaca peripheral lymphocytes, designing two pairs of degenerate primers, amplifying variable region (VHH) gene segments of heavy chain antibody immunoglobulin subtypes IgG2 and IgG3 naturally existing in two alpacas bodies through PCR, recombining in phagemids, converting into inhibition type E.coli competent cells, adding auxiliary phage, reassembling, and secreting out of bodies to form a phage display nano antibody library.

The screening of the phage display nanobody library can adopt a solid phase screening method. The solid phase screening method is that antigen target molecules are fixed on a solid phase medium (an immune tube, an enzyme label plate and the like), phage display nano antibody libraries are added for full binding reaction, then nonspecific and low-affinity phage are washed and removed, specific and high-affinity phage are eluted and recovered, and the phage are amplified in E.coli and then used for next round of panning. Through several rounds of adsorption-elution-enrichment biopanning methods, the phage display nano antibody specifically binding with the target molecule can be screened out.

Because the nano antibody of the small molecular substance is prepared by using the complete antigen of coupling the small molecule and the carrier protein, in alpaca serum, not only antibodies aiming at the small molecule but also antibodies aiming at the carrier protein exist, and because the antigenic determinants of the carrier protein are numerous, the number of the antibodies of the carrier protein in the alpaca serum is far more than that of the small molecule antibodies. In the corresponding phage display nano antibody library, the nano antibodies aiming at carrier proteins are far more than the nano antibodies aiming at small molecules, and the screening of the small molecule nano antibodies in the library can be seriously interfered by the carrier protein nano antibodies with a large number. Therefore, how to remove the nano antibody of the carrier protein efficiently in the screening process of the small molecule substance nano antibody has important significance for screening the small molecule nano antibody.

Disclosure of Invention

The invention provides a method for removing phage expressing carrier protein nano-antibody in a hapten phage display nano-antibody library, which comprises the following steps of (1) incubating magnetic beads coupled with carrier protein and the hapten phage display nano-antibody library to obtain magnetic beads combined with phage expressing carrier protein nano-antibody; (2) and removing the magnetic beads of the phage combined with the expression carrier protein nano antibody by adopting magnetic separation, thereby removing the phage expressing the carrier protein nano antibody in the hapten phage display nano antibody library.

Optionally, according to the method, the magnetic beads are magnetic beads with carboxyl groups on the surface.

Optionally, according to the above method, in the magnetic beads coupled with the carrier protein, the mass ratio of the total carboxyl groups of the magnetic beads to the carrier protein is 20: 1 to 5: 1.

Optionally, the method repeats steps (1) - (2)1 or more times, such as 3-6 times, according to the method described above.

Optionally, the carrier protein is Bovine Serum Albumin (BSA), Ovalbumin (OVA), human serum albumin (HAS), or hemocyanin (KLH) according to the method described above. In particular, the method can be based on the coupling protein of the complete antigen used for small molecule substance immunization.

Alternatively, the incubation conditions for step (1) are 25-37 ℃, e.g. 25 ℃, the reaction time is 2-3 hours, e.g. 3 hours, the pH is 7.0-9.0, e.g. pH7.4, according to the method described above.

Alternatively, the hapten phage-display nanobody library is prepared according to the above-described method using camelids or sharks.

Optionally, according to the above method, the magnetic beads coupled with the carrier protein are obtained by reacting the magnetic beads and the carrier protein after gently mixing them uniformly for 3 hours, during which the suspension state of the magnetic beads is maintained.

The application of the method in the preparation of hapten nano-antibodies also belongs to the protection scope of the invention.

As above, the hapten can be a small molecule that does not contain amino acids.

The method provided by the invention carries out chemical coupling on carrier protein and magnetic beads with carboxyl, incubates the constructed phage display nano antibody library and the magnetic beads coupled with the carrier protein, the phage expressing the carrier protein nano antibody in the library can be combined with the carrier protein coupled on the surface of the magnetic beads, and a part of phage expressing the carrier protein nano antibody in the library can be removed by magnetically separating the magnetic beads combined with the phage expressing the carrier protein nano antibody. Repeating the steps for 3-6 times by using the new carrier protein magnetic beads, so that the quantity of phage for expressing the carrier protein nano antibody in the small molecule nano antibody library can be greatly reduced, and the method provides help for subsequent efficient screening of the target small molecule nano antibody.

The magnetic bead coupled with the carrier protein can effectively remove the phage expressing the carrier protein nano antibody in the small molecule phage display nano antibody library, namely reversely screen out unnecessary phage expressing the carrier protein nano antibody from the small molecule substance phage display nano antibody library, thereby improving the screening efficiency of the small molecule nano antibody.

Drawings

FIG. 1 shows the synthetic route of atrazine hapten.

FIG. 2 shows the synthetic route of atrazine small molecule complete antigen.

FIG. 3 shows the result of the magnetic bead-coupled carrier protein assay of example 1, wherein the left side shows the test result of the magnetic bead-coupled carrier protein assay, and the right side shows the OD of the test result_450nmReading the graph.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The data were processed using EXCEL software and the results are expressed as mean values.

The microplate reader used in the following examples was purchased from Thermo corporation.

The test materials used in the following examples were prepared by purchasing mouse anti-Bovine Serum Albumin (BSA) monoclonal antibody from Baiolaibo, horseradish peroxidase-labeled anti-M13 murine monoclonal antibody from Doctorka Biochemical Co., Ltd., carboxyl magnetic beads (cat # 70102-50, average particle diameter 2 μ M, concentration 10mg/ml) from Suzhou beaver biomedical engineering Co., Ltd., and other reagents were purchased from conventional biochemical reagent stores, unless otherwise specified.

The immunogen used below was prepared as follows:

1. synthesis of small-molecule hapten of atrazine

1) 7.0g (38.04m mol) of cyanuric chloride was dissolved in 40mL of Tetrahydrofuran (THF), and the reaction was cooled to-10 ℃. 4.48g (75.93m mol) of isopropylamine are dissolved in 15ml of THF, the isopropylamine solution is slowly added dropwise to the cyanuric chloride solution, the reaction system is warmed to room temperature and stirred for 3 hours, and the completion of the reaction of the starting materials is monitored by thin-layer chromatography TLC (P/E5: 1). Most of THF was removed by concentration under reduced pressure, the reaction was partitioned with ethyl acetate/water, the organic phase was collected and washed with a small amount of 0.1N HCl, and the organic phase was dried and concentrated and recrystallized from petroleum ether to give 6.8g of a white solid with a yield of 86.4%.

2)1.0g of the compound H1a was dissolved in 15mL of THF, 0.386g (9.65m mol) of NaOH and 0.497g of aminobutyric acid (4.83m mol) were dissolved in 8mL of water and added dropwise to the reaction system at room temperature. After dripping, stirring the reaction system at room temperature for 5 hours, detecting by TLC (P/E1: 1) that the raw materials react completely, concentrating under reduced pressure to remove most THF, adjusting the pH of the reaction system to acidity by using 6N HCl, generating a large amount of white solid, pulping the white solid obtained by suction filtration by using 7mL absolute ethyl alcohol (EtOH) for 5 hours, and obtaining 0.81g of white solid by suction filtration, wherein the yield is 61.3 percent, and the white solid is the atrazine Hapten (Hapten).

The synthetic route of the atrazine hapten is shown in figure 1.

2. Synthesis of atrazine small molecule complete antigen

1) Synthesis of atrazine-BSA complete antigen

Dissolving 5.5mg (20 mu mol) of atrazine hapten in 0.6mL of Dimethylformamide (DMF), adding 12.4mg (60 mu mol) of Dicyclohexylcarbodiimide (DCC) and 6.9mg (60 mu mol) of N-hydroxysuccinimide (NHS), and stirring at room temperature for 18-24 hours to obtain solution A; dissolving 30mg BSA in 3.0ml PBS (0.01M, pH7.4) buffer solution to obtain solution B, adding the solution A into solution B, and stirring at 4 deg.C for 10-12 hr to obtain solution C; and (3) putting the solution C into a dialysis bag, dialyzing in PBS (0.01M, pH7.4) buffer solution, changing the solution every 3 hours for 6 times, collecting the solution in the dialysis bag, subpackaging, and refrigerating at-20 ℃ to obtain the atrazine-BSA complete antigen.

2) Synthesis of atrazine-OVA complete antigen

The synthesis of atrazine-OVA complete antigen needs to couple atrazine hapten and OVA, and the specific operation process refers to the atrazine-BSA complete antigen synthesis method. The specific dosage is different, the dosage of OVA is 10mg, the dosage of hapten is 1.87mg, the dosage of DCC is 1.4mg, and the dosage of NHS is 0.78 mg.

The synthetic route of the atrazine small molecule complete antigen is shown in figure 2.

The method for preparing the atrazine phage display nano antibody library used in the following examples is as follows:

1. animal immunization

200ug of the prepared atrazine-BSA complete antigen was taken, and the primary immunization was carried out by mixing with complete Freund's adjuvant in equal volume, stirring overnight with a magnetic stirrer at 4 ℃ and emulsifying. The three-week old male alpaca was injected with multiple injections into the neck and back, and immunized once every two weeks. The adjuvant used for emulsification from the second immunization is changed into Freund incomplete adjuvant. Blood is collected for 1ml before each immunization from six immunizations and is used for detecting the alpaca serum titer. Selecting the highest titer once, and taking blood and building a library after 7-10 days.

2. Total RNA extraction

Blood is collected 7-10 days after primary immunization with the highest titer and is used for bank building, and total RNA in alpaca blood is separated by adopting a LeukoLOCK total RNA extraction kit of Life Technology company according to an operation manual.

Synthesis of first Strand of cDNA

First strand cDNA was synthesized from the above-mentioned total RNA in alpaca blood using Invitrogen's reverse transcription kit

4. Amplification of heavy chain antibody variable region VHH genes

Taking respective degenerate primer pairs of the IgG2 and IgG3 variable region VHH genes, respectively carrying out Polymerase Chain Reaction (PCR) amplification by taking cDNA as a template, wherein the primer pair F, R2 is used for cloning an IgG2 subtype, and the primer pair F, R1 is used for cloning an IgG3 subtype.

Primer and method for producing the same

K is G or T;

primer R1:

primer R2:

the reaction system is as follows:

10xPCR buffer 5. mu.L

50mM MgSO₄ 2μL

10mM dNTP 1μL

10 μ M primer F1 μ L

10 μ M primer R2/R11 μ L

DNA polymerase 0.2. mu.L

cDNA 2μL

H₂O 37.8μL

Total 50 μ L

The PCR procedure was:

pre-denaturation at 94 ℃ for 2min

30 cycles: 30s at 94 ℃; 30s at 55 ℃; 1min at 68 DEG C

68℃ 5min

4℃∞

The PCR reaction system is 50 mu L of one reaction, and the existing content ratio of immunoglobulin IgG2 and IgG3 in the camelidae is 3: 2, so that the VHH gene for amplifying IgG2 during library construction is 6 PCR reactions, and the PCR reaction system for the VHH gene for IgG3 is 4. After the PCR reaction was completed, the products in each PCR tube were analyzed by agarose gel electrophoresis.

5. Gel recovery of heavy chain antibody variable region VHH gene PCR products

The amplified product of the VHH gene was gel-recovered using a gel recovery kit from QIAGEN.

Enzyme digestion of VHH Gene and vector

The primer for amplifying the VHH gene is provided with an SfiI restriction site, and the phagemid vector pComb3X (Biovector Inc. Biovector113372) is also provided with 2 SfiI restriction sites with different sequences, so that the PCR product of the VHH gene and the DNA of the vector pComb3X plasmid are subjected to enzyme digestion by Sfi I restriction endonuclease to obtain a cohesive end, and then subsequent ligation reaction can be carried out to obtain a recombinant plasmid.

Plasmid DNA pComb3X and VHH fragment DNA were digested with SfiI endonuclease (20U/. mu.L) from NEB corporation, respectively. The adding amount of SfiI endonuclease in the enzyme digestion reaction system depends on the quality of the added DNA, and 36U of SfiI restriction endonuclease is added into each mu g of VHH gene DNA; 6U sfiI restriction enzyme was added per μ g of pComb3X plasmid DNA; add 10 Xbuffer and 100 XBSA, and finally ddH according to the total volume of the reaction₂And (4) fixing the volume by using O. The digestion conditions are 50 ℃ for 16 h.

Directly purifying the enzyme digestion product of the VHH gene by using a PCR purification kit of QIAGEN company, and then measuring the concentration of the purified product by using Nanodrop; and (3) recovering the carrier fragment by gel electrophoresis after the pComb3X plasmid enzyme digestion reaction is finished, quickly cutting the carrier fragment with the size of about 3400bp under an ultraviolet lamp after the electrophoresis is finished, purifying the carrier fragment by a gel recovery kit, and finally detecting the gel recovery effect and concentration by electrophoresis.

Ligation of VHH Gene fragments to vectors

T4 ligase of NEB company is selected to connect the enzyme digestion product of the VHH gene with the enzyme digestion fragment of the pComb3X vector, the connection molar ratio of the pComb3X vector to the VHH gene is 1: 3, and the reaction system is as follows:

10 Xbuffer 20. mu.L

VHH fragment cleavage product 0.495 ug

pComb3X enzyme digestion product 1.4. mu.g

Add ddH₂O to a total volume of 200. mu.L

The ligation was carried out at 16 ℃ overnight,

8. recombinant phagemid electrotransfer

(1) Purifying the ligation product by using a PCR purification kit, and finally eluting by using 30 mu L of sterile deionized water;

(2) adding 3 mu L of the purified ligation product into 25 mu L of E.coli ER2738 competent cells, slightly mixing, completely sucking out and transferring to a precooling electric rotating cup (the inner diameter is 1mm), quickly placing in an electric rotating instrument for electric conversion, wherein the conditions of electric conversion are as follows: 200 Ω, 18KV, 25 μ F;

(3) immediately adding 975 mu L of 37 ℃ preheated SOC culture medium into an electric transfer cup after electric shock, gently sucking and uniformly mixing by using a liquid transfer gun, transferring into a bacteria shaking tube, and performing shake recovery culture for 1h at 250rpm in a 37 ℃ shaking table;

(4) the electric transfer was repeated 10 times, each time 3. mu.L of the ligation product, 10 times of the transformed bacterial solution were pooled, 1uL of the pooled bacterial solution was diluted 10-fold with sterile water and spread on an LB-Amp plate, and cultured overnight in an incubator at 37 ℃.

9. Library capacity and diversity identification of Nanobody libraries

Calculating the library capacity by counting the number of monoclones in an overnight cultured LB-ampicillin (LB-ampicillin) plate, wherein the total clone number of 10mL of converting bacteria is the library of the established nano antibody libraryCapacity, library capacity ═ clone number x10⁵。

20 single clones were randomly picked from LB-Amp plates, inoculated into 1mL of SB liquid medium, added with ampicillin to a final concentration of 50. mu.g/mL, shake-cultured overnight at 37 ℃ and 250rpm, and sent to the sequencer the next day for sequencing, using a gack primer as a sequencing primer.

10. Construction of phage display Nanobody library

(1) The whole recovered and cultured bacterial solution was transferred to 200mL SB medium, 200. mu.L of 50mg/mL ampicillin and 200. mu.L of 20mg/mL tetracycline were added, and shake culture was performed at 37 ℃ and 250rpm until OD was reached₆₀₀About 0.6;

(2) adding 1mL of M13KO7 helper phage, and standing at 37 ℃ for 30 mm;

(3) culturing at 250rpm and 37 deg.C for 2h with shaking, adding 200 μ L70mg/mL kanamycin, and culturing at 37 deg.C at 250rpm overnight;

(4) the next day, the bacterial liquid is subpackaged in a centrifugal bottle, centrifuged at 10000rpm for 15min, and the supernatant is collected and called phage supernatant for short:

(5) transferring the phage supernatant to a sterile 500mL centrifugal bottle, adding a quarter volume of PEG/NaCl solution, uniformly mixing, placing on ice, and standing for 2h to precipitate phage;

(6) centrifuging at 10000rpm for 20min at 4 deg.C, discarding the supernatant, and adding 10mL PBS buffer (containing 1 Xprotease inhibitor, 0.5% BSA, 0.02% NAN)₃) Resuspending the pellet;

(7) filtering the resuspended phage solution with a 0.22 μm sterile filter, collecting the filtered and sterilized phage solution as the atrazine phage display nano antibody library, which is also called as original phage display nano antibody library hereinafter. The atrazine phage display nano antibody library contains phage expressing nano antibody resisting atrazine and phage expressing nano antibody resisting carrier protein.

Example 1 removal of phage expressing Nanobodies of Carrier proteins from libraries Using Carrier protein-coupled magnetic beads

Activation of carboxyl groups on the surface of magnetic beads

1. Putting 100 μ l of the mixed carboxyl magnetic beads into a 1.5ml centrifuge tube, placing the centrifuge tube on a magnetic separation rack, carrying out magnetic separation to remove supernatant, washing for 2 times by using 200 μ l of MEST solution (100mM MES, pH5.0, 0.05% Tween20, solvent is water), and then carrying out magnetic separation to remove supernatant;

2. rapidly adding 100 μ l of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) solution (10mg/mL, using the MEST solution as a dispersant) and 100 μ l N-hydroxysuccinimide (NHS) solution (10mg/mL, using the MEST solution as a dispersant) into a centrifuge tube containing magnetic beads, vortexing to fully suspend the magnetic beads, activating at 25 ℃ for 30 minutes, and performing reverse mixing by using a vertical mixer during the vortexing to maintain the suspended state of the magnetic beads; after the steps, the carboxyl on the surface of the magnetic bead is activated, and can be coupled with the carrier protein with amino. The activated state cannot be preserved for a long time and the coupling should be carried out immediately.

Coupling of magnetic beads and carrier proteins

1. Placing the centrifuge tube on a magnetic separation frame, performing magnetic separation to remove supernatant, adding 200 μ g of bovine serum albumin (BSA (carrier protein)), optimizing the appropriate amount and concentration according to specific experiments, keeping the pH of the solution about 8.0, and simultaneously adding 0.05% Tween20 to improve the dispersibility of magnetic beads and avoid reagents containing amino groups except the carrier protein in a buffer system, and gently mixing the mixture;

2. coupling for 3 hours at 25 ℃, and keeping the suspension state of the magnetic beads during the coupling;

3. the well-coupled centrifuge tube with magnetic beads is placed on a magnetic separation frame for magnetic separation to remove supernatant, and 200. mu.l of PBST solution (pH7.2) is added for resuspension of the magnetic beads.

4. The supernatant was removed by magnetic separation, washed 3 times with 200. mu.l each of PBS (pH7.2), resuspended in PBS (pH7.2) to obtain carrier protein-coupled magnetic beads, and stored at 4 ℃.

Thirdly, checking the coupling effect of the magnetic beads and the carrier protein

1. Respectively putting 200 mu l of magnetic bead 1 tube which is not coupled with carrier protein and magnetic bead 2 tube which is coupled with carrier protein into a 1.5ml centrifuge tube, wherein the magnetic bead which is not coupled with carrier protein is used as a blank control and is marked as CK; the other two tubes are magnetic beads coupled with carrier protein and respectively marked as BSA and 0;

2. adding 100 μ l PBS solution into CK tube and 0 tube, adding 100 μ l PBS solution with BSA concentration of 1mg/ml into BSA tube, adding 100 μ l mouse anti-Bovine Serum Albumin (BSA) monoclonal antibody into the three tubes, and incubating at 37 deg.C for 0.5 hr;

3. after the incubation is finished, washing the magnetic beads for 4 times by using 1% PBST, then respectively adding 200 mu l of horse radish peroxidase labeled goat anti-mouse IgG into each tube, and incubating for 0.5 hour at 37 ℃;

4. after the incubation is finished, washing the magnetic beads for 4 times by using 1% PBST, then adding 200 mu l of 3, 3 ', 5, 5' -tetramethyl benzidine color development solution into each tube, and developing for 10 minutes in a dark place;

5. finally, 100. mu.l of 1M hydrochloric acid is added to terminate the reaction, and the OD is read by an enzyme-linked immunosorbent assay_450nm。

The results of the experiment are shown in FIG. 3.

The results of the experiments show that the OD of the blank well (CK)_450nmRead 0.347, OD of control well (0)_450nmReading 4.183, OD of inhibited well (BSA)_450nmReading 1.354. The difference in absorbance between blank well (CK) and control well (0) indicates that BSA has been coupled to the beads. There is also a difference in absorbance values between the control well (0) and the suppression well (BSA), indicating that BSA coupled to the magnetic beads and the added BSA standard can compete for anti-Bovine Serum Albumin (BSA) monoclonal antibodies, further indicating that the magnetic beads have been coupled to carrier proteins.

Fourthly, removing bacteriophage of the expression carrier protein nano antibody in the library by adopting magnetic beads coupled with the carrier protein

1. Putting 200 mul of magnetic beads coupled with carrier protein into a 1.5ml centrifuge tube, then putting the centrifuge tube on a magnetic separation frame for magnetic separation to remove preservation solution, then adding 1ml of constructed atrazine phage display nano antibody library into the centrifuge tube, incubating for 30 minutes at room temperature, reversing and uniformly mixing the library by using a vertical mixer during incubation, and keeping the suspension state of the magnetic beads. The first incubation was completed and the supernatant was collected by magnetic separation and was the library completed by incubation.

2. And after the first incubation is finished, taking 100 mu l of incubated library for monitoring the condition that the carrier protein nano antibody is removed from the library, then adding the rest incubated libraries into the next centrifugal tube filled with new coupled carrier protein magnetic beads, incubating for 30 minutes again, magnetically separating and collecting supernate, and repeating the incubation for 6 times in sequence. The removal effect of the phage expressing the carrier protein nano antibody in the library is detected by enzyme-linked immunosorbent assay (ELISA) for the library after each incubation.

The enzyme-linked immunosorbent assay (ELISA) method comprises the following steps:

1. 1. mu.g/ml of the carrier protein BSA was prepared in coating buffer (0.05mol/L carbonate buffer, pH 9.6), coated on an ELISA plate at 100. mu.l/well, and incubated at 37 ℃ for 3 hours.

2. The supernatant was discarded and the plate was washed 4 times with 1% PBST.

3. The original phage display nano antibody library and the library of phage with 6 rounds of incubation and removal of the expression vector protein nano antibody are diluted by 100 times by PBST, added into an enzyme label plate, 100 mu l/hole and incubated for 1 hour at 37 ℃.

4. The supernatant was discarded and the plate was washed 4 times with 1% PBST.

5. Horseradish peroxidase-labeled anti-M13 murine monoclonal antibody (HRP) (1: 10000) was added to the cells at 100. mu.l/well and incubated at 37 ℃ for 1 hour.

6. The supernatant was discarded and the plate was washed 4 times with 1% PBST.

7. Adding 3, 3 ', 5, 5' -tetramethyl benzidine color developing solution, 100 μ l/hole, and developing for 10 minutes in dark.

8. The reaction was stopped by adding 1M hydrochloric acid, 50. mu.l/well, OD read by microplate reader_450nm。

The results are shown in Table 1.

TABLE 1 incubation of Carrier protein magnetic beads with library detection of Carrier protein antibodies in the library

The experimental result shows that the atrazine phage display nano antibody library and the magnetic bead coupled with the carrier proteinAfter six incubation rounds, OD_450nmThe values decreased gradually, indicating that the phage expressing the carrier protein nanobody decreased in the library in turn. The magnetic beads coupled with the carrier protein provided by the invention are proved to have obvious effect on removing the phage of the expression carrier protein nano antibody in the small molecule phage display nano antibody library.

Example 2 Small molecule Nanobody screening

Single, small molecule nano antibody screening process

Screening of small molecule nano antibody incubated by magnetic beads

1. Incubating the atrazine phage display nano antibody library and the magnetic beads coupled with the carrier protein prepared in the embodiment 1 for 6 times at room temperature, and performing the incubation according to the fourth step in the embodiment 1 to obtain an incubated library;

2. preparing 1 mu g/ml atrazine-OVA coated enzyme label plate by using a coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6), coating the plate at 4 ℃ overnight, discarding the supernatant coated overnight in the enzyme label plate the next day, and washing the plate for 3 times by using 0.1% PBST;

3. adding the incubated library of step 1 diluted 10 times with phosphate buffer (pH7.4, 0.01mol/L) to the wells coated with atrazine-OVA, incubating for 2 hours at 37 ℃, and then washing the plate 10 times with 1% PBST;

4. adding 100 mul of 1000ng/ml atrazine standard solution, and shaking to react for 1 hour at room temperature;

5. collecting supernatant as eluate, and adding 2ml OD into the eluate_600nm0.6-1.0 of ER2738 Escherichia coli, standing at 37 ℃ for 30 minutes, then supplementing 6mL of SB culture medium (wherein the final concentration of ampicillin is 50 mug/mL), and shaking at 37 ℃ and 220rpm for 2 hours;

6. then, 1ml of helper phage M13KO7 was added, and 91ml of SB medium (in which final concentration of ampicillin was 50. mu.g/ml) was supplemented, and after 2 hours of culture at 37 ℃, kanamycin (final concentration was 70. mu.g/ml) was added, and the mixture was cultured overnight;

7. centrifuging at 1000rpm for 15 minutes the next day, and taking the supernatant (the supernatant is the amplified bacteriophage expressing the atrazine nano antibody, and is hereinafter referred to as the supernatant of the step 7) to perform an indirect enzyme-linked immunosorbent assay.

(II) screening of small molecule nano antibody without magnetic bead incubation

1. Preparing 1 mu g/ml atrazine-OVA coated enzyme label plate by using a coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6), coating the plate at 4 ℃ overnight, discarding the supernatant coated overnight in the enzyme label plate the next day, and washing the plate for 3 times by using 0.1% PBST;

2. adding an original phage display nano antibody library diluted by 10 times by using phosphate buffer (pH7.4, 0.01mol/L) into the wells coated with the atrazine-OVA, incubating for 2 hours at 37 ℃, and then washing the plate for 10 times by using 1% PBST;

3. adding 100 mul of 1000ng/ml atrazine standard solution, and shaking to react for 1 hour at room temperature;

4. collecting supernatant as eluate, and adding 2ml OD into the eluate_600nm0.6-1.0 of ER2738 Escherichia coli, standing at 37 ℃ for 30 minutes, then supplementing 6mL of SB culture medium (wherein the final concentration of ampicillin is 50 mug/mL), and shaking at 37 ℃ and 220rpm for 2 hours;

5. then, 1ml of helper phage M13KO7 was added, and 91ml of SB medium (in which final concentration of ampicillin was 50. mu.g/ml) was supplemented, and after 2 hours of culture at 37 ℃, kanamycin (final concentration was 70. mu.g/ml) was added, and the mixture was cultured overnight;

6. centrifuging at 1000rpm for 15 minutes the next day, and taking the supernatant (the supernatant is the amplified bacteriophage expressing the atrazine nano antibody, and is hereinafter referred to as the supernatant of the step 8) to perform an indirect enzyme-linked immunosorbent assay.

Second, indirect enzyme-linked immunosorbent assay of supernatant

The treatment groups were divided into 4 treatment groups, control group, inhibition group, OVA group and BSA group.

1. Preparing a reaction hole of the enzyme labeling plate coated with 1 mu g/ml atrazine-OVA by using a coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6) in a control group, incubating for 3 hours at 37 ℃ in 100 mu L/hole; the inhibition group uses coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6) to prepare 1 mu g/ml atrazine-OVA coating reaction hole of the ELISA plate, 100 mu L/hole, incubate for 3 hours at 37 ℃; OVA group was incubated at 37 ℃ for 3 hours in a reaction well of an ELISA plate coated with 1. mu.g/ml OVA (ovalbumin) prepared with a coating buffer (0.05mol/L carbonate buffer, pH 9.6); the BSA group was incubated with 1. mu.g/ml of the carrier protein BSA in coating buffer (0.05mol/L carbonate buffer, pH 9.6) at 37 ℃ for 3 hours in reaction wells of an ELISA plate coated with 1. mu.g/ml of BSA.

2. Plates were washed 4 times with 1% PBST.

3. Mu.l of phosphate buffer (pH7.4, 0.01mol/L) was added to each reaction well of the OVA group, the BSA group and the control group, 50. mu.l of phosphate buffer (pH7.4, 0.01mol/L) containing 1000ng/ml atrazine standard was added to each reaction well of the inhibition group, and the supernatant of step 7 and the supernatant of step 8 prepared above were added to each reaction well of the control group, the inhibition group, the OVA group and the BSA group, 50. mu.l/well, and incubated at 37 ℃ for 1 hour.

4. The supernatant was discarded and the plates were washed 4 times with 1% PBST.

5. Mu.l of horseradish peroxidase-labeled mouse anti-M13 antibody (HRP) (1: 10000) was added to each reaction well and incubated at 37 ℃ for 1 hour.

6. The supernatant was discarded and the plates were washed 4 times with 1% PBST.

The results are shown in Table 2.

TABLE 2 supernatant detection (absorbance OD)_450nm)

Note: the supernatant obtained in the step 7 is the supernatant obtained after competitive screening and amplification of the antibody library without carrier protein; and the supernatant obtained in the step 8 is the supernatant obtained after competitive screening and amplification of the antibody library without the carrier protein.

The experimental result shows that the light absorption values of a control group, a suppression group and a BSA group of the supernatant of the library after incubation, competitive screening and amplification of the carrier protein magnetic beads and the light absorption values of the non-carrier protein magnetic beadsAnd the difference between the absorbance values of all groups of the library supernatant after incubation, competitive screening and amplification is obvious. The color development of the BSA group decreased from 3.122 to 1.011, indicating that the amount of carrier protein antibodies in the supernatant decreased greatly after panning and amplification of the library treated with the carrier protein magnetic beads, further demonstrating the effect of removing the carrier protein antibodies in example 1. The color rendering value of the corresponding OVA group is also reduced from 1.784 to 0.529. Inhibition rate ((control OD)_450nmInhibition group OD_450nm) Control group OD_450nm) From 11.30% to 22.20%. The relative content of the small molecule nano antibody in the treated supernatant is higher. The removal of the carrier protein nano antibody in the supernatant is beneficial to the subsequent screening of the atrazine nano antibody.

Third, positive clone identification of library

1. 20 clone colonies (i.e., colonies on a plate coated after the gradient dilution of escherichia coli treated by 1-5 in the step (I) and colonies on a plate coated after the gradient dilution of escherichia coli treated by 1-4 in the step (II)) are respectively and randomly selected from a library titer plate after the competitive screening of an atrazine phage display nano antibody library which is incubated by magnetic beads and not incubated by magnetic beads, the clone colonies are inoculated in 3ml of SB culture medium for activation, 3 mul of 100mg/ml ampicillin solution is added, the mixture is cultured at 37 ℃ and 220rpm until the OD600nm is 0.6-0.8, 10 mul of auxiliary phage M13KO7 is respectively added, the mixture is kept standing for 30 minutes, the shaking culture is continued for 2 hours, 3 mul of 70mg/ml of kanamycin solution is added, and the mixture is cultured at 37 ℃ and 220rpm overnight. The next day, centrifugation was carried out at 4 ℃ and 10000rpm for 5 minutes, and then the supernatant was taken for use.

2. The treatment groups were divided into 4 treatment groups, control group, inhibition group, OVA group and BSA group. Preparing a reaction hole of the enzyme labeling plate coated with 1 mu g/ml atrazine-OVA by using a coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6) in a control group, incubating for 3 hours at 37 ℃ in 100 mu L/hole; the inhibition group uses coating buffer solution (0.05mol/L carbonate buffer solution, pH is 9.6) to prepare 1 mu g/ml atrazine-OVA coating reaction hole of the ELISA plate, 100 mu L/hole, incubate for 3 hours at 37 ℃; OVA group uses coating buffer solution (0.05mol/L carbonate buffer solution, pH 9.6) to prepare 1 mug/ml reaction hole of OVA coated enzyme label plate, 100 mug/hole, incubate 3 hours at 37 ℃; the BSA group was incubated with 1. mu.g/ml of the carrier protein BSA in coating buffer (0.05mol/L carbonate buffer, pH 9.6) at 37 ℃ for 3 hours in reaction wells of an ELISA plate coated with 1. mu.g/ml of BSA.

3. The supernatant was discarded and the plates were washed 4 times with 1% PBST.

4. Mu.l of phosphate buffer (pH7.4, 0.01mol/L) was added to each reaction well of the OVA group, the BSA group and the control group, and 50. mu.l of phosphate buffer (pH7.4, 0.01mol/L) containing 1000ng/ml atrazine standard was added to each reaction well of the inhibition group. The supernatant obtained in step 1 was added to the reaction wells of the control group, the inhibition group, the OVA group and the BSA group, 50. mu.l/well and incubated at 37 ℃ for 1 hour.

5. The supernatant was discarded and the plates were washed 4 times with 1% PBST.

6. Horseradish peroxidase-labeled mouse anti-M13 antibody (HRP) (1: 10000) was added at 100. mu.l/well and incubated at 37 ℃ for 1 hour.

7. The supernatant was discarded and the plates were washed 4 times with 1% PBST.

8. Adding 3, 3 ', 5, 5' -tetramethyl benzidine color developing solution, 100 μ l/hole, and developing for 10 minutes in dark.

9. The reaction was stopped by adding 1M hydrochloric acid, 50. mu.l/well and OD read_450nmThe number of monoclonals inhibiting atrazine was counted.

The results are shown in Table 3.

TABLE 3 identification of monoclonals picked from competitive screen titer plates following incubation and non-incubation with vector protein magnetic beads

The experimental result shows that the number of the monoclonals (namely the atrazine nano antibody) showing inhibition in 20 randomly picked clones on the library titer plate after the incubation of the carrier protein and the competitive screening is 16, and accounts for 80 percent; the number of inhibited monoclonals in 20 randomly picked clones on the library titer plate without carrier protein magnetic bead incubation and competitive screening was 5, accounting for 25%. The result shows that after the carrier protein nano antibody is removed through the incubation of the carrier protein magnetic beads, the screening efficiency of the subsequent small molecule nano antibody can be greatly improved, and the screening is more favorable for obtaining the target small molecule nano antibody. The removal of the carrier protein nano antibody in the small-molecule phage nano antibody library reduces the interference of the carrier protein antibody in the screening process, is beneficial to screening to obtain the small-molecule nano antibody, and improves the screening efficiency.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. The method for removing the phage expressing the carrier protein nano antibody in the hapten phage display nano antibody library is characterized in that: comprises that

(1) Incubating the magnetic beads coupled with the carrier protein and the hapten phage display nano-antibody library to obtain magnetic beads combined with phage of the expression carrier protein nano-antibody;

(2) and removing the magnetic beads of the phage combined with the expression carrier protein nano antibody by adopting magnetic separation, thereby removing the phage expressing the carrier protein nano antibody in the hapten phage display nano antibody library.

2. The method of claim 1, wherein: the magnetic beads are magnetic beads with carboxyl on the surface.

3. The method of claim 2, wherein: in the magnetic beads coupled with the carrier protein, the mass ratio of the total carboxyl of the magnetic beads to the carrier protein is 20: 1 to 5: 1.

4. The method according to any one of claims 1-3, wherein: the method repeats the steps (1) - (2) more than 1 time.

5. The method according to any one of claims 1-4, wherein: the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

6. The method according to any one of claims 1-5, wherein: the incubation condition of the step (1) is 25-37 ℃, the reaction time is 2-3 hours, and the pH value is 7.0-9.0.

7. The method according to any one of claims 1-6, wherein: preparing the hapten phage display nano antibody library by adopting camelid or shark.

8. Use of the method of any one of claims 1-7 for the preparation of hapten nanobodies.