Nanobody targeting pancreatic polypeptide, coding sequence and its application

Abstract

The present invention relates to the field of bioengineering technology, in particular to a nanobody targeting pancreatic polypeptide, a coding sequence thereof and an application thereof. The nanobody provided by the present invention is a single-domain antibody obtained by immunizing alpacas with pancreatic polypeptide as an immunogen and then screening through library construction, which can specifically bind pancreatic polypeptide in serum and can detect pancreatic polypeptide in the sera of chickens, sheep and humans by ELISA method.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a nanobody targeting pancreatic polypeptide, a coding sequence thereof, and applications thereof. Background Art

[0002] A specific antibody that is naturally lacking in heavy chains and has biological activity in camelids (alpacas, camels) and cartilaginous fish is called a single-domain antibody. The antigen-binding site (VHH) of the single-domain antibody has an independent antigen recognition ability, and the independently expressed VHH is also called a nanobody. Compared with traditional tetramer antibodies, the main characteristics of single-domain antibodies are: small molecular weight, simple structure, stable physical and chemical properties, etc. The excellent properties of nanobodies make them have advantages in many aspects: in terms of the antibody entering the body, nanobodies can cross some protective barriers in the animal body and enter the diseased site to play a role, such as the blood-brain barrier, blood-testis barrier, etc.; in terms of antigen-antibody binding, they can bind to some hidden antigen epitopes, especially suitable for targets where it is difficult to obtain antibodies, such as GPCRs, ion channels, and enzyme active centers, etc.; in terms of reducing production costs, the structure of nanobodies is simple and easy to express in vitro, and at the same time, inclusion bodies are not easily produced during in vitro expression, and the production process is simple; at the same time, the small molecular weight and simple structure of nanobodies are more conducive to genetic modification and humanization modification of nanobodies.

[0003] Pancreatic polypeptide (PP) is a by-product discovered when purifying insulin from the pancreas. The functional region of pancreatic polypeptide in mammals is a linear peptide composed of 36 amino acids, and its structure is conservative, with only 1-2 amino acid differences in mammals. In the normal pancreas, F cells in the islets of Langerhans in its peripheral region secrete pancreatic polypeptide, which is a member of the neuropeptide Y (NPY) family. As one of the biomarkers, pancreatic polypeptide is of great significance for the auxiliary diagnosis of pancreatic neuroendocrine tumors. However, there is currently a lack of single-domain antibodies with good specificity for pancreatic polypeptide. Summary of the Invention

[0004] To solve the above problems, the present invention provides a nanobody targeting pancreatic polypeptide, a coding sequence thereof, and applications thereof. The nanobody provided by the present invention is a single-domain antibody screened from alpacas and has good specificity for pancreatic polypeptide antigen.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a nanobody targeting pancreatic polypeptide, and its amino acid sequence is as shown in SEQ ID NO.5.

[0007] The present invention provides the coding sequence of the nanobody described in the above technical solution, and the nucleotide sequence of the coding sequence is shown in SEQ ID NO.14.

[0008] The present invention provides an expression vector containing the coding sequence described in the above technical solution.

[0009] Preferably, the original vector of the expression vector includes the pET28a vector.

[0010] The present invention provides an engineered bacterium containing the expression vector described in the above technical solution.

[0011] Preferably, the original strain of the engineered bacterium includes Escherichia coli.

[0012] Preferably, the Escherichia coli includes Escherichia coli BL21(DE3).

[0013] The present invention provides the application of the nanobody described in the above technical solution, or the coding sequence described in the above technical solution, or the expression vector described in the above technical solution, or the engineered bacterium described in the above technical solution in the preparation of a product for detecting pancreatic polypeptide.

[0014] Preferably, the product includes a reagent or kit for detecting by ELISA method.

[0015] Beneficial effects:

[0016] The present invention provides a nanobody targeting pancreatic polypeptide, and the amino acid sequence is shown in SEQ ID NO.5. The nanobody provided by the present invention is a single-domain antibody obtained by immunizing alpaca with pancreatic polypeptide as an immunogen and screening through library construction, which can specifically bind pancreatic polypeptide in serum and can detect pancreatic polypeptide in chicken, sheep and human sera by ELISA method. Brief description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 It is the SDS-PAGE identification result of step (c) in Example 1;

[0019] Figure 2 It is the SDS-PAGE identification result of the purified sample in step (d) of Example 1;

[0020] Figure 3 It is the SDS-PAGE identification result of the sample after enzymatic digestion in step (d) of Example 1;

[0021] Figure 4It is the gel electrophoresis diagram of the first-round PCR-VHH;

[0022] Figure 5 It is the electrophoresis diagram of the second-round PCR-VHH;

[0023] Figure 6 It is the result of detecting the library capacity of the pancreatic polypeptide nano-library by plate;

[0024] Figure 7 It is the result of detecting the abundance of the pancreatic polypeptide nano-library by plate;

[0025] Figure 8 It is the result of detecting the insertion rate of the pancreatic polypeptide library by PCR;

[0026] Figure 9 It is the result of detecting the purified pancreatic polypeptide nanobody by SDS-PAGE;

[0027] Figure 10 It is the ELISA detection result of three kinds of nanobodies against pancreatic polypeptide;

[0028] Figure 11 It is the result of detecting the purified pancreatic polypeptide nanobody by Western Blot;

[0029] Figure 12 It is the result of detecting the content of pancreatic polypeptide in animal and human sera by ELISA. Specific implementation mode

[0030] The present invention provides a nanobody targeting pancreatic polypeptide, and its amino acid sequence is shown as SEQ ID NO.5.

[0031] The present invention provides the coding sequence of the nanobody described in the above technical solution. The nucleotide sequence of the coding sequence is as shown in SEQ ID NO.14, specifically as follows: 5'-GAGTCTGGAGGGGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGGCCCTTCAGTATCTATGCCATGGGCTGGTACCGCCAGGCGCCAGGGAAGCAGCGCGAGTTGGTTGCAGCTATTACTAGTGGTGGGAGCACAAACTATGCAGACTCCGTGAAGGGTCGATTTACGATCTCCAGAGACAACGCCAAGAACATGGTGTATCTGCAAATGGACAGCCTGAAACCCGAGGACACGGCCGTCTATTACTGTAATGCAGACCCCCCCTTAGGAGATACCGACTATGACGACTATGCGTACTGGGGCCAGGGGACCGACGTCACCGTCTCCTCA-3'.

[0032] The present invention provides an expression vector containing the coding sequence described in the above technical solution. In the present invention, the original vector of the expression vector preferably includes the pET28a vector; the coding sequence is preferably located between the BamHⅠ and SalⅠ restriction enzyme sites of the pET28a vector. When constructing the expression vector, the preparation method of the coding sequence preferably includes artificial synthesis or PCR amplification; the primers for PCR amplification are preferably as shown in SEQ ID No.8 and SEQ ID No.9; the PCR amplification preferably includes the artificially synthesized coding sequence or the glycerol bacteria of the clone strain with correct sequencing results in the examples.

[0033] The present invention provides an engineered bacterium containing the expression vector described in the above technical solution. In the present invention, the original strain of the engineered bacterium preferably includes Escherichia coli; the Escherichia coli preferably includes Escherichia coli BL21(DE3).

[0034] The present invention provides the application of the nanobody described in the above technical solution, or the coding sequence described in the above technical solution, or the expression vector described in the above technical solution, or the engineered bacterium described in the above technical solution in the preparation of a product for detecting pancreatic polypeptide. In the present invention, the product preferably includes reagents or kits for detection by ELISA method. The pancreatic polypeptide nanobody provided by the present invention has binding properties with pancreatic polypeptide in animal and human sera, and has good sensitivity and affinity.

[0035] To further illustrate the present invention, the nanobody targeting pancreatic polypeptide, its coding sequence and their applications provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

[0037] Preparation of immunogen:

[0038] (1) Download the nucleotide sequence corresponding to pancreatic polypeptide (GeneBank accession number: NM_001319209.2) according to the NCBI database, insert the pancreatic polypeptide sequence into the plasmid pSumo-Mut with a fusion tag N-His-Sumo to obtain the pSumo-Mut-pancreatic polypeptide recombinant plasmid; the pancreatic polypeptide sequence is as shown in SEQ ID NO.15, specifically 5'-GCTCCCCTAGAACCAGTATATCCTGGAGATAACGCGACCCCGGAACAAATGGCACAGTATGCGGCGGACCTGCGTCGTTACATCAATATGTTGACCCGTCCGCGCTACGGCAAGCGCCACAAAGAAGATACGCTCGCGTTCAGCGAGTGGGGTAGCCCACATGCTGCCGTTCCGCGTGAGCTGTCCCCGCTGGACTAA-3'; the pancreatic polypeptide sequence is located after the ggc base of the His-Sumo tag of the plasmid pSumo-Mut.

[0039] (2) Transform the pSumo-Mut-pancreatic polypeptide recombinant plasmid and the pSumo-Mut plasmid into the BL21(DE3) strain respectively and obtain the corresponding pancreatic polypeptide expression strains for induction expression and protein purification. The specific method is as follows:

[0040] (a) Incubate the transformed and plated bacterial solution overnight, pick the monoclonal colonies on the culture plate the next day and incubate them overnight; preserve the overnight cultured bacterial solution to obtain glycerol bacteria.

[0041] (b) Inoculate the glycerol bacteria into the TB medium containing Kana (final concentration of 50 ng / μl) resistance according to a volume ratio of 1:1000, shake and culture overnight at 37°C; transfer the overnight cultured bacterial solution to 1 L of fresh TB medium according to a volume ratio of 1:100 the next day, shake and culture at 37°C and 200 rpm until OD 600 = 0.8, add IPTG with a final concentration of 0.2 mM, induce overnight at 15°C and 120 rpm, and set an uninduced control group at the same time.

[0042] (c) Collect the overnight-induced bacterial solution and centrifuge it at 7000×g for 10 min to obtain the bacterial cells; resuspend and wash the cells twice with PBS, and then use an ultrasonic disruptor to disrupt them (300 W, 30 min); centrifuge at 20000×g at 4 °C for 1 h. Collect the supernatant after centrifugation, take a small portion for SDS-PAGE identification, and the results are shown in Figure 1 , where M: protein molecular weight standard, 1: pSumo-mut induction (empty vector), 2: non-induced, 3: induced, 4: supernatant after induction and disruption, 5: precipitate after induction and disruption; the rest is stored at -80 °C for later use.

[0043] (d) Purify the obtained supernatant by affinity chromatography using a pre-packed nickel column, and identify the protein purity and concentration of the purified sample by SDS-PAGE. The results are shown in Figure 2 , where M: protein molecular weight standard, 1: processed sample after disruption, 2: liquid flowing out after the sample passes through the nickel column, 3 - 5: eluent. Cut off the tag of the purified protein by enzymatic cleavage (Sumo protease), and then detect whether the tag is successfully removed by passing through the nickel column again. The results are shown in Figure 3 , where M: protein molecular weight standard, 1: protein before enzymatic cleavage, 2: protein after enzymatic cleavage, 3: liquid flowing out after incubation with Ni-IDA packing after enzymatic cleavage. Use the protein after enzymatic cleavage as an immunogen for subsequent library construction.

[0044] Example 2

[0045] The construction method of the alpaca-derived anti-pancreatic polypeptide nanobody library is as follows:

[0046] 1. Prepare a formulation by mixing the immunogen prepared in Example 1 and adjuvant at a volume ratio of 1:1. Emulsify the obtained formulation and immunize alpacas subcutaneously at multiple points at 200 μg / time for four times, with a two-week interval between each immunization. Except for the complete Freund's adjuvant used in the first immunization, the incomplete Freund's adjuvant is used in other immunizations;

[0047] 2. Collect 50 ml of anticoagulated alpaca blood 1 week after the 4th immunization, isolate lymphocytes (2.04×10 8 cells), extract the total RNA of lymphocytes, and reverse transcribe the RNA into cDNA. The specific method is as follows:

[0048] Mix 5 μg of total RNA, 1 μl of Random6 Primer, 1 μl of Oligo(dT) 18Mix the primer, 1 μl of dNTP Mix and 7 μl of ddH2O, and process the mixture at 65 °C for 5 min. Let the obtained processed product stand on ice for 5 min. Then mix the standing product with 4 μl of 5×PrimeScript II buffer, 0.5 μl of RNase inhibiter, 1 μl of Primer Scrip II Reverse Transcriptase and 4.5 μl of ddH2O, and successively process at 40 °C for 40 min and 70 °C for 15 min to obtain cDNA;

[0049] 3. Using the cDNA described in step 2 as a template, perform the first round of PCR amplification with Call001-F and Call002-R primers. Among them, Call001-F corresponds to the Leader region of the alpaca antibody, and Call002-R corresponds to the second constant region (CH2) of the alpaca antibody, and can respectively perform PCR amplification on the leader region and CH2 region of the conventional antibody (900 bp) and the heavy chain antibody (700 bp); The reverse transcription product is divided into 30 reactions, and the primer sequences are as follows:

[0050] Call001-F: 5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID No.1;

[0051] Call001-R: 5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID No.2;

[0052] The reaction system of the PCR amplification is: 2 μl of cDNA, 1 μl of Call001-F with a concentration of 10 μmol / μl, 1 μl of Call001-R with a concentration of 10 μmol / μl, 25 μl of Taq Green PCR Mix (ComWin Biotech) and 21 μl of ddH2O; The reaction program is: 95 °C for 5 min; 95 °C for 30 s, 53 °C for 30 s, 72 °C for 40 s, 30 cycles; 72 °C for 5 min;

[0053] 4. Agarose gel electrophoresis analysis shows that the products of the first round of PCR reaction mainly contain two kinds of amplification products of the conventional antibody (900 bp) and the heavy chain antibody (700 bp) (see Figure 4 ). Cut the gel and recover the nucleic acid of 700 bp under ultraviolet light. Use a gel recovery kit (ComWin Biotech) to recover and purify the nucleic acid fragment of 700 bp. Connect 1 μl of the gel recovery product to the pMD-19T vector to obtain a ligation product; The ligation system is: 1 μl of pMD-19T vector, 1 μl of gel recovery product, 5 μl of Solution I, and make up to 10 μl with ddH2O; The reaction condition is: react overnight at 4 °C;

[0054] 5. Mix the ligation product with DH5α competent cells, incubate on ice for 25 min, perform precise heat shock at 42 °C for 90 s, incubate on ice for 4 min, add 400 μl of LB medium, incubate at 37 °C and 200 rpm for 40 min, spread 50 μl of the transformed bacterial solution evenly on the surface of LB solid medium containing ampicillin (AMP, final concentration 100 μg / mL), invert the culture plate and culture overnight at 37 °C; the next day, pick 20 monoclonal colonies and inoculate them into 5 ml of LB liquid medium with AMP resistance for overnight culture, and then determine their base sequences;

[0055] 6. According to the sequencing results, design the second-round PCR primers VHH2-F and VHH2-R for library construction at the constant regions FR1 and FR4 at both ends of the alpaca single-domain antibody (VHH domain) respectively, and perform a total of 24 PCR reactions. The primer sequences are as follows:

[0056] VHH2-F: 5'-TTTCTATTACTAGGCCCAGCCGGCCGAGTCTGGAGGRRGC TTGGTGCA-3', SEQ ID No. 3;

[0057] VHH2-R: 5'-AAACCGTTGGCCATAATGGCCTGAGGAGACGRTGACSTS GGTC-3', SEQ ID No. 4;

[0058] The reaction system for the second-round PCR is as follows: 6 μl of 700 bp amplification product with a concentration of 800 μg / μl, 2 μl of VHH2-F with a concentration of 10 μmol / μl, 2 μl of VHH2-R with a concentration of 10 μmol / μl, 25 μl of Taq Green PCR Mix, and 15 μl of ddH2O; the reaction program is: 95 °C for 5 min; 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, 30 cycles; 72 °C for 5 min;

[0059] 7. Take 2 μl of the second-round PCR reaction product for agarose gel electrophoresis detection (see Figure 5 ) and purify it according to the nucleic acid purification kit instruction manual. The size of the amplification product (VHH fragment) is about 400 bp, the band is single, and the size is consistent with the expectation;

[0060] 8. The amplified products obtained in step 7 and the pCANTAB5e vector (Beijing Baokewei Food Safety Biotechnology Co., Ltd.) were respectively subjected to a second round of digestion to eliminate the empty vector rate. The enzyme used in the reaction was Sfi I (Thermo); for the first round of digestion, both the VHH and the vector were digested in 20 reactions. The reaction conditions were: digestion in a water bath at 50 °C for 1 h; the VHH system was purified using a PCR product purification kit; the vector digestion system was first purified by agarose and then purified using a gel extraction kit; the purified products after the first round of digestion were again incubated in a water bath at 50 °C and digested for 1 h, and the two products were purified again.

[0061] 9. Ligation and purification of the digested products: The VHH fragment and the vector pCANTAB5e after two rounds of digestion and purification were ligated. T4 ligase (Thermo) was used for the ligation. The reaction system was: 1 μg of VHH fragment, 3 μg of pCANTAB5e vector, 10 μl of T4 DNA Ligase, 20 μl of 10× Buffer, and ddH2O was added to make up to 200 μl; the reaction system was vortexed instantaneously and reacted overnight at 4 °C. The next day, the ligation product was purified according to the instructions.

[0062] 10. Construction and characterization of the pancreatic polypeptide nanobody library: The purified pCANTAB5e-VHH ligation product (total volume 100 μl) was added to freshly prepared TG1 electrocompetent cells, pipetted and mixed well, and incubated on ice for 10 min; under the conditions of voltage 1.8 KV, resistance 200 Ω, capacitance 25 μF, and time 5 ms, after transformation in an electroporator, the TG1 cells after electroporation in the electroporation cup were transferred to a 50 ml sterile centrifuge tube and incubated with shaking at 37 °C for 1 h; the incubated bacterial solution was centrifuged at 5000 g for 5 min, the supernatant was discarded completely, and the pellet was resuspended by adding 8 ml of fresh SOC medium; 100 μl of the bacterial solution was serially diluted and then spread on 16 2×YTAG solid culture plates (90 mm) to determine the library capacity. Two plates were used for each dilution, and a total of 8 dilution plates were made. The remaining bacterial solution was spread on 30 2×YTAG solid culture plates (150 mm plates) and cultured overnight in a static incubator at 25 °C; the next day, after the colonies on the culture plates grew well, 5 ml of 2×YT liquid medium was added to each of the 39 150 mm plates to wash the colonies and collect the washing solution; centrifuged at 5000 g at 4 °C for 12 min, the pellet was resuspended by adding 90 ml of 2×YT liquid medium containing 15% glycerol, and after sealing in separate tubes, it was the prepared primary library bacteria, named pancreatic polypeptide nanobody library. 100 μl of the primary library bacteria was taken to determine the library abundance, and the rest was aliquoted and stored frozen at -70 °C.

[0063] 11. Determination of the library capacity: 100 μl of the electroporated bacterial solution was serially diluted, and the dilution factor ranged from 10 -1 ~10 -8; Take 100 μl of each dilution and coat 2 2×YTAG solid culture plates, and culture overnight at 25°C; The next day, count the colonies on the gradient plate and calculate the library capacity. The results are shown in Figure 6 , where there are 3 monoclonal colonies on the 10 -6 dilution plate, and the library capacity is 3÷(100×10 -6 )×30×10 3 = 9×10 8 ;

[0064] 12. Determination of library abundance: Take 100 μl of the primary library bacterial liquid for gradient dilution, with dilution factors from 10 -4 to 10 -10 ; Take 100 μl of the bacterial liquid of each dilution and coat 2 2×YTAG solid culture plates, and culture overnight at 25°C; The next day, count the colonies on the gradient plate ( Figure 7 ), calculate the library abundance. The abundance of the library is 50÷(100×10 -10 )×10 3 = 5×10 12 CFU / ml;

[0065] 13. Determination of the insertion rate and insertion diversity of the VHH fragment in the library: After determining the library capacity, randomly pick 60 monoclonal colonies from the solid culture plate with the determined library capacity and inoculate them into 1 ml of 2×YTAG medium, and culture overnight at 37°C with shaking; The next day, perform bacterial liquid PCR identification on 60 clones, and send the remaining bacterial liquid to a sequencing company for sequencing; Analyze the PCR identification results and sequencing results, and calculate the insertion rate and insertion diversity of the VHH fragment.

[0066] All 60 bacterial liquid PCR samples meet the VHH fragment length ( Figure 8 ). Only 2 clones in the 60 sequencing samples have unqualified sequencing results and are not VHH fragments. NCBI analysis of the sequences of the qualified sequencing samples found that they are all VHH sequences. Therefore, the insertion rate of the VHH fragment in the library can be calculated as 100%, and the sequence diversity is 97%. Software analyzes the amino acid sequences corresponding to the 58 sample sequences and analyzes their structures. It can be seen that the pancreatic polypeptide nanobody library constructed in the present invention is divided into a constant region and a variable region with obvious partitions. The above all indicate that the pancreatic polypeptide nanobody library constructed in the present invention is an immune library with good characteristics and is suitable for screening specific nanobodies.

[0067] Example 3

[0068] Perform the first round of panning on the pancreatic polypeptide nanobody library prepared in Example 2 to obtain pancreatic polypeptide-VHH1, and aliquot and store it frozen at -70°C.

[0069] During elution, 50 mM sodium carbonate / sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg / ml, the coating volume was 2 ml, and the immunization tube was coated with pancreatic polypeptide.

[0070] The elution method is as follows:

[0071] 1) Inoculate 500 μl of the pancreatic polypeptide nanobody library prepared in Example 2 into 100 ml of 2×YTAG medium, and incubate with shaking at 37 °C and 200 rmp for 1 hour until the OD 600 reaches 0.4;

[0072] 2) Add KM13 helper phage. Add 100 μl of KM13 helper phage to 100 ml of the bacterial solution, let it stand and infect at 37 °C for 30 minutes, and then incubate with shaking for 30 minutes;

[0073] 3) Centrifuge at 4000×g for 10 minutes to remove the culture medium supernatant, resuspend the bacterial cell precipitate with 100 ml of 2×YTAK medium, and incubate with shaking at 30 °C and 200 rmp overnight;

[0074] 4) The next morning, centrifuge the overnight culture at 11000×g and 4 °C for 10 minutes, transfer the supernatant to a new centrifuge bottle and add 20 ml of PEG / NaCl solution, mix well and incubate in an ice bath for 70 minutes;

[0075] 5) Centrifuge at 11000×g and 4 °C for 30 minutes, discard the supernatant, and then centrifuge again for 2 minutes to completely aspirate the supernatant;

[0076] 6) Resuspend the precipitate with 2.6 ml of PBS buffer, and then aliquot it into 2 1.5-ml centrifuge tubes, and centrifuge at 11600×g for 10 minutes;

[0077] 7) Recover the supernatant, name it ZJ-pancreatic polypeptide-VHH1, take 100 μl for titer determination, and mix the remaining with 1.6 ml of MPBS solution and incubate at room temperature for 1 h to obtain a mixed solution (phage supernatant treated with MPBS solution) for later use.

[0078] Coated protein treatment:

[0079] (1) The day after coating the protein, pour out the liquid in the immunization tube and wash the tube 3 times with PBS buffer.

[0080] (2) Fill each tube with MPBS, block at room temperature for 2 h, and then wash the tube 3 times with PBS buffer.

[0081] (3) Add 2 ml of the mixed solution obtained in step 7) above to the immunization tube, incubate at room temperature for 2 h, then wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer.

[0082] (4) Add 2 ml of 100 mM TEA solution to each tube, gently shake at room temperature for 15 min to elute the bound phages, and then add 2 ml of Tris-HCl solution to neutralize.

[0083] (5) Transfer the eluted phages (named XT-Pancreatic Polypeptide-VHH1) to a 50-ml centrifuge tube, and add 16 ml of TG1 bacterial solution with an OD 600 of 0.4. Incubate in a 37 °C water bath for 30 minutes to allow the eluted phages to infect the TG1 bacterial solution, and add 4 ml of TG1 bacterial solution with an OD 600 of 0.4 to the immunization tube for infection. Finally, combine them, with a total volume of 24 ml.

[0084] (6) Take 100 μl of the bacterial solution for titer determination, and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0085] (7) Resuspend the bacterial pellet with 1 ml of 2×YT medium, and spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150-mm plates), and incubate overnight in a 30 °C incubator.

[0086] (8) The next day, collect the colonies grown on the plates with 2×YT medium, add glycerol to a final concentration of 15% at 60%, which is the primary library bacteria, named Pancreatic Polypeptide-VHH1, aliquot and store frozen at -70 °C.

[0087] Determine the titer of the rescued phages: Gradient dilute ZJ-Pancreatic Polypeptide-VHH1, with dilution factors from 10 -7 to 10 -13 ; Take 10 μl of phages from each dilution to infect 190 μl of TG1 bacterial solution with an OD 600 of 0.4; Take 100 μl of the bacterial solution from each dilution and spread it on a 2×YTAG solid culture plate, and incubate overnight in a 30 °C incubator; Count the colonies on the assay plate and calculate the titer of ZJ-Pancreatic Polypeptide-VHH1 (denoted as ZJ).

[0088] Determine the titer of the eluted phages: Gradient dilute XT-Pancreatic Polypeptide-VHH1, with dilution factors from 10 -1 to 10 -5 ; Take 100 μl of the bacterial solution from each dilution and spread it on a 2×YTAG solid culture plate, and incubate overnight in a 30 °C incubator; Count the colonies on the assay plate and calculate the titer of XT-Pancreatic Polypeptide-VHH1 (denoted as XT); Furthermore, calculate the input-output ratio ZJ / XT of the first round of panning.

[0089] On the basis of the first round of panning, perform the second to fourth rounds of panning in sequence: The coating concentrations of pancreatic polypeptide are 10 μg / ml, 5 μg / ml, and 5 μg / ml respectively; The dilution factors for determining the titer of the rescued phages are 10-7 ~10 -12 、10 -8 ~10 -11 、10 -8 ~10 -11 ; The dilution factors for measuring the eluted phage titer were 10 -1 ~10 -6 、10 -1 ~10 -6 、10 -1 ~10 -6 ; After the eluted phage was neutralized with Tris-HCl solution (1 M, pH 7.4), 200 μl of the phage was taken to infect 800 μl of TG1 bacterial solution with an OD 600 of 0.4 (100 μl was taken for serial dilution, and the remaining was used for bacterial preservation), and then 10 -3 ~10 -6 A total of 4 dilution factors were made. For each dilution factor, 3 2×YTAG solid culture plates (150 mm plates) were coated, with 100 μl of the bacterial solution on each plate, and cultured overnight at 30 °C; the colonies on the culture plates were counted, the titer was calculated, and the culture plates were labeled as plates and stored in a 4 °C refrigerator for later use.

[0090] Screening of specific nanobodies:

[0091] Preparation of monoclonal phage supernatant: 192 monoclonal strains were picked from each plate and inoculated into 2 96-well deep-well culture plates, with 200 μl of 2×YTAG medium in each well. The culture plates were labeled as PP-1 and PP-2 respectively and cultured with shaking at 30 °C. After 8 h, 20 μl of the bacterial solution was taken from each well and inoculated into 180 μl of 2×YTAG medium, and cultured with shaking at 37 °C. 60 μl of 60% glycerol was added to the remaining bacterial solution in the original plate to a final concentration of 15%, and stored at -80 °C. After 1 h of culturing the transferred plates with shaking, 20 μl of the mixture of KM13 and YTAG (60 μl of KM13 + 12 ml of 2×YTAG) helper phage was added to each well, and allowed to infect statically at 37 °C for 30 min, and then cultured with shaking at 37 °C for 40 min. The deep-well plates were centrifuged at 1800×g for 10 min, the supernatant was discarded, and 400 μl of 2×YTAK medium was added to each well to resuspend the precipitate, and cultured with shaking at 30 °C overnight. The next day, centrifuged at a maximum speed of 2020×g for 20 min, 250 μl of the phage supernatant was aspirated from each well and transferred to a new deep-well plate, and 250 μl of the blocking solution (PBS buffer solution containing 3% BSA) was added to each well and incubated at room temperature for 1 h, to be used for indirect ELISA detection.

[0092] Identification of specific monoclonal phages:

[0093] The reactivity of the phage supernatant with pancreatic polypeptide was detected by indirect ELISA as follows: A 96-well ELISA plate was coated with pancreatic polypeptide at a concentration of 2 μg / ml, 100 μl per well, and incubated overnight at 4°C. The next day, the coating liquid in the wells was discarded (Solarbio, C1050), and 100 μl of blocking solution (3% BSA + PBS) was added to each well and blocked at 37°C for 1 h. The blocking solution in the wells was discarded, and 100 μl of the phage supernatant obtained from the four rounds of screening treated with the blocking solution was added to each well as the primary antibody and incubated at 37°C for 1 h. The plate was washed 12 times with PBST washing solution. 100 μl of the secondary antibody (HRP-M13 Antibody, dilution 1:10,000) was added to each well and incubated at 37°C for 1 h. The plate was washed 12 times with PBST washing solution. 100 μl of the chromogenic substrate (TMB chromogenic solution, Solarbio, PR1200) was added to each well, and the reaction was carried out in the dark for 5 - 15 min. Then, 100 μl of the termination solution (Solarbio, C1050) was added to each well to terminate the reaction. The 96-well ELISA plate was placed on a microplate reader to read the OD 450 absorbance value. The ELISA results were analyzed and the positive well numbers were determined. At the same time, PBS was set as the negative control.

[0094] The reactivity of the phage supernatants corresponding to 192 monoclonal antibodies with pancreatic polypeptide was detected by indirect ELISA. Twenty monoclonal antibodies with good reactivity with pancreatic polypeptide were selected according to the results of the indirect ELISA test, and the results are shown in Table 1.

[0095] Table 1 ELISA screening results of pancreatic polypeptide monoclonal antibodies (OD 450 absorbance value)

[0096]

[0097]

[0098] The cultured bacterial solutions of the 20 monoclonal antibodies were sent to a sequencing company for sequencing. The amino acid sequences obtained by sequencing and prediction (merging duplicates) were as follows:

[0099] The amino acid sequence of the Nb1 pancreatic polypeptide nanobody is shown in SEQ ID NO.5, specifically: ESGGGLVQPGGSLRLSCAASGRPFSIYAMGWYRQAPGKQRELVAAITSGGST NYADSVKGRFTISRDNAKNMVYLQMDSLKPEDTAVYYCNADPPLGDTDYDD YAYWGQGTDVTVSS;

[0100] The amino acid sequence of the Nb2 pancreatic polypeptide nanobody is shown in SEQ ID NO.6, specifically: ESGGGLVQAGGSLRLSCAASGFTFDEYVIGWFRQAPGKEREGISCISSSDGIAY YADSVSGRFTISTDIAKSTVYLQMDSLKPEDTAVYYCAKDRGMWGGYDYWG QGTEVTVSS;

[0101] The amino acid sequence of the Nb3 pancreatic polypeptide nanobody is shown in SEQ ID NO.7, specifically: SLVQPGGSLRLSCTASDFALQDQTIGYFRQIPGKEREGVSCISTREQSTYYADS VKGRFTIGRDNANNAVYLQMNSLKPEDSAVYYCAADLSGGCRSWHRPSVRY GMDYWGKGTHVIVSS.

[0102] Example 4

[0103] Activity and affinity of pancreatic polypeptide nanobody

[0104] Construction of prokaryotic expression recombinant plasmid: Inoculate the glycerol bacteria of the clone strain with correct sequencing results in Example 3 into 5 ml of 2×YTAG medium for culture, and extract the plasmid using a plasmid miniprep kit as the template plasmid for prokaryotic expression (or an artificially synthesized nucleotide sequence can also be used as the template). Then design primers for prokaryotic expression, and introduce BamHⅠ and SalⅠ restriction enzyme sites at the 5' end and 3' end of the primers respectively. Amplify the nanobody VHH sequence using the designed primers, and ligate it into the pET28a prokaryotic expression vector through the above restriction enzyme sites to construct a prokaryotic expression recombinant plasmid of the nanobody for the specific identification of pancreatic polypeptide of the nanobody.

[0105] Primers for prokaryotic expression:

[0106] Nb1-F: 5'-CGGATCCGAGTCTGGAGGGGGCTTGG-3', SEQ ID No.8;

[0107] Nb1-R: 5'-GCGTCGACTGAGGAGACGGTGACGTCG-3', SEQ ID No.9;

[0108] Nb2-F: 5'-CGGATCC GAGTCTGGAGGAGGCTTGGT-3', SEQ ID No.10;

[0109] Nb2 - R: 5'-GCGTCGACTGAGGAGACGGTGACCTCG-3', SEQ ID No.11;

[0110] Nb3 - F: 5'-CGGATCCGAGTCTGGAGGAGGCTTGGT-3', SEQ ID No.12;

[0111] Nb3 - R: 5'-GCGTCGACTGAGGAGACGATGACGTGGG-3', SEQ IDNo.13.

[0112] The screening steps are as follows: The recombinant plasmid and the pET28a empty vector are transformed into the BL21(DE3) strain to obtain the corresponding nanobody expression strains. Then, the nanobodies are induced to express. The specific method is:

[0113] The bacterial liquid after transformation and plating is cultured overnight. The next day, single colonies on the culture plate are picked and cultured overnight. The bacterial liquid cultured the next day is used for glycerol preservation of bacteria.

[0114] The next day, 50 μl of the bacterial liquid is taken and inoculated into 5 ml of LB medium with Kan + resistance, and 2 tubes are inoculated respectively. The culture is shaken at 37 °C until the OD 600 reaches 0.6; IPTG is added to one tube of the bacterial liquid for induction (final concentration 0.8 mM), and the other tube is not added with IPTG as an uninduced control. The culture is shaken at 15 °C overnight; at the same time, a BL21(DE3) empty strain control is made, and the empty strain control is cultured using LB medium without resistance.

[0115] The SDS - PAGE identification of the nanobody is as follows:

[0116] 1 ml of the induced and uninduced cultured bacterial liquid is respectively taken into 1.5 ml centrifuge tubes, and centrifuged at 13000 rpm for 2 min; the supernatant is discarded, and the bacterial cell precipitate is washed 2 times with PBS buffer; the bacterial cell precipitate is resuspended with 20 μl of PBS buffer, then 5 μl of 5× protein loading buffer is added, and the sample is boiled in boiling water for 5 minutes. The sample is electrophoresed on a 15% polyacrylamide gel. After the electrophoresis is completed, the gel is stained with Coomassie Brilliant Blue staining solution for 1 h, and then decolorized with decolorizing solution. The results are shown in Figure 9 . From Figure 9 it can be seen that when the imidazole concentration is 75 nM, the target protein is successfully eluted, and the collected protein band is single, indicating high purity and can be used for subsequent experiments.

[0117] Screening of nanobodies with anti - pancreatic polypeptide neutralizing activity: The glycerol strains corresponding to the nanobodies Nb1, Nb2, and Nb3 screened in Example 3 are respectively inoculated into 5 ml of Kan +LB medium with resistance, after shaking culture at 37°C for 10 h, transfer it to 500 ml of Kan + LB medium with resistance, shake culture at 37°C until 600 When OD reaches 0.6, add IPTG (final concentration 0.8 mM) to induce expression, and shake culture overnight at 15°C. The next day, purify the three kinds of nanobodies expressed by the above three glycerol bacteria.

[0118] Identification of the purified product: Coat pancreatic polypeptide protein at a concentration of 2 μg / ml, 100 μL / well, and use the coating solution as a blank control at the same time, and place it at 4°C overnight. The next day, discard the coating liquid in the wells, add 100 μL of blocking solution (3% BSA) to each well and block at 37°C for 1 h. Discard the blocking solution in the wells, add Nb1, Nb2, and Nb3 diluted with PBS to each well respectively, and use PBS as a negative control, and incubate at 37°C for 1 h. Wash the plate 10 times with PBST washing solution, add 100 μL of His-tag antibody (1:15000) to each well, and incubate at 37°C for 1 h. Wash the plate 10 times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, and react in the dark for 10 min. Then add 100 μL / well of ELISA termination solution to terminate the reaction. Subsequently, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD 450 nm value, and finally take P (positive) / N (negative) ≥ 2.1 as positive. The results are shown in Figure 10 .

[0119] It can be seen from Figure 10 that the pancreatic polypeptide nanobody with the best affinity is Nb1, and this antibody is identified with His-tag by Western Blotting method: after SDS-PAGE electrophoresis, transfer it to the NC membrane, directly label it with His secondary antibody, and display the antibody by developing technique. The results are shown in Figure 11 . It can be seen from Figure 11 that there is a His-positive imprinting band, and the molecular weight is about 17 kDa, which is consistent with the size of the nanobody.

[0120] Application of pancreatic polypeptide nanobody: Coat different chicken, sheep and human sera by ELISA, use the purified pancreatic polypeptide nanobody Nb1 as the primary antibody, and His-tag antibody (purchased from Kangwei Century, product number CW0285) as the secondary antibody for labeling and color development to show pancreatic polypeptide in the serum. The specific method is as follows:

[0121] Coat chicken serum, sheep serum and human serum with ELISA coating solution at a ratio of 1:1, 100 μL / well, and use the coating solution as a blank control at the same time, and place it at 4°C overnight.

[0122] The next day, discard the coating liquid in the wells, add 100 μL of blocking solution (3% BSA) to each well, and block at 37°C for 1 h. Discard the blocking solution in the wells, add Nb1 diluted with PBS to each well, use PBS as a negative control, and incubate at 37°C for 1 h. Wash the plate 10 times with PBST washing solution, add 100 μL of His-tag antibody (1:15000) to each well, and incubate at 37°C for 1 h. Wash the plate 10 times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, and react in the dark for 10 min. Then add 100 μL / well of ELISA stop solution to terminate the reaction. Subsequently, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD 450 nm value, and finally, a positive result is defined as P (positive) / N (negative) ≥ 2.1. The results are shown in Figure 12 .

[0123] Through ELISA detection, it was found that there were positive reactions for pancreatic polypeptide in the chicken serum, sheep serum, and human serum to be detected, indicating that the purified nanobody of pancreatic polypeptide can be used for the detection of pancreatic polypeptide in serum.

[0124] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Nanobody targeting pancreatic polypeptide, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

5.

2. The coding molecule of the nanobody according to claim 1, characterized in that, The nucleotide sequence of the encoding molecule is as shown in SEQ ID NO.

14.

3. An expression vector containing the encoding molecule according to claim 2.

4. The expression vector according to claim 3, wherein, The original vector of the expression vector includes the pET28a vector.

5. A genetically engineered bacterium containing the expression vector according to claim 3 or 4.

6. The engineered bacterium according to claim 5, characterized in that, The original strain of the engineered bacterium includes Escherichia coli ( Escherichia coli ).

7. The engineered bacterium according to claim 6, wherein The Escherichia coli includes Escherichia coli BL21(DE3).

8. Use of the nanobody according to claim 1, the encoding molecule according to claim 2, the expression vector according to claim 3 or 4, or the genetically engineered bacterium according to any one of claims 5 to 7 in the preparation of a product for detecting pancreatic polypeptide.

9. The application according to claim 8, wherein The product includes reagents or kits for detection by ELISA method.

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