A recombinant plasmid nanobody mutant M1 and a preparation method and application thereof

Abstract

The application discloses a recombinant plasmid nanobody mutant M1 and a preparation method and application thereof, and aims to provide the recombinant plasmid nanobody mutant M1 which has high affinity to an antigen, high sensitivity and high specificity when detecting HCC. The amino acid sequence of the recombinant plasmid nanobody mutant M1 is shown as SEQ ID NO. 2, and the application belongs to the technical field of biotechnology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a recombinant plasmid nanobody mutant M1 and a preparation method and application thereof. BACKGROUND

[0002] Hepatocellular carcinoma is the most common primary cancer of the liver, including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (cHCC-CCA), of which HCC accounts for about 90% of all primary liver cancer. The current diagnostic markers for HCC include alpha-fetoprotein, alpha-fetoprotein isoform L3, abnormal prothrombin and glypican 3 (GPC3).

[0003] The GPC3 value in the serum of liver cancer patients is usually higher than that of cirrhosis patients and the like. For small mass liver cancer (<3cm), the GPC3 positive rate is relatively high, reaching 86.67%, and in alpha-fetoprotein (AFP) negative liver cancer patients, the GPC3 positive rate is 75%. Therefore, GPC3 as a specific marker for HCC is expected to become an important target for tumor visualization precise diagnosis and treatment.

[0004] The detection antibodies for GPC3 protein include polyclonal antibodies, monoclonal antibodies and nanobodies. The latest nanobodies combine the beneficial properties of monoclonal antibodies and small molecule drugs compared with traditional antibodies. The antibodies targeting specific antigens are usually obtained by screening from an antibody library. After obtaining the antibodies, mutations are performed to generate a new antibody library, and then antibodies with increased affinity are screened from the mutant library. Although this iterative method can effectively screen antibodies with higher binding force, it is time-consuming and expensive. Obviously, using a directed mutation strategy can help researchers obtain high-affinity antibodies more cheaply and conveniently. SUMMARY

[0005] In view of the above problems, the present application aims to provide a recombinant plasmid nanobody mutant M1, which has strong affinity with antigens, high sensitivity and strong specificity when detecting HCC.

[0006] The second object of the present application is to provide a preparation method of the recombinant plasmid nanobody mutant M1, which is time-saving.

[0007] To this end, the first technical solution provided by the present application is as follows:

[0008] The application provides a recombinant plasmid nanobody mutant M1, and an amino acid sequence of the recombinant plasmid nanobody mutant M1 is shown as SEQ ID NO. 2.

[0009] The application provides a second technical scheme of the application, and the application provides a preparation method of the recombinant plasmid nanobody mutant M1.

[0010] Further, the preparation method of the recombinant plasmid nanobody mutant M1, and an amino acid sequence of the anti-GPC3 nanobody NB-MG18 is shown as SEQ ID NO. 1.

[0011] Further, the preparation method of the recombinant plasmid nanobody mutant M1, and an amino acid sequence of the anti-GPC3 nanobody NB-MG18 is shown as SEQ ID NO. 1.

[0012] Further, the preparation method of the recombinant plasmid nanobody mutant M1,

[0013] When the HIS30 is mutated into ASN30 and the PRO28 is mutated into PHE28, a forward primer F1 nucleotide sequence used is shown as SEQ ID NO. 3, and a reverse primer R1 nucleotide sequence used is shown as SEQ ID NO. 4.

[0014] When the LEU101 is mutated into GLN101, a forward primer F2 nucleotide sequence used is shown as SEQ ID NO. 5, and a reverse primer R1 nucleotide sequence used is shown as SEQ ID NO. 6.

[0015] Further, the preparation method of the recombinant plasmid nanobody mutant M1, and the gene site-directed mutation kit used is a QuickMutation gene site-directed mutation kit.

[0016] Further, the preparation method of the recombinant plasmid nanobody mutant M1, and the gene site-directed mutation kit used is a QuickMutation gene site-directed mutation kit.

[0017] 1) Homology modeling

[0018] The anti-GPC3 nanobody is used as an initial sequence, a three-dimensional structure of the nanobody is constructed by using a homology modeling technology, and a homologous antibody with a sequence similarity of 100% is selected as a template after SAVES evaluation;

[0019] 2) Molecular docking

[0020] Using the HDCOK program to perform protein-protein docking, taking the anti-GPC3 nanobody NB-MG18 as the receptor and the GPC3 protein as the ligand to perform molecular docking to predict the structure of the antigen-antibody complex formed by the anti-GPC3 nanobody NB-MG18 and the GPC3 protein;

[0021] 3) Analysis of key amino acids of docking

[0022] Using the online website InterProSurf and PyMOL for analysis and prediction of amino acid residues of the nanobody located at the interaction interface with the GPC3 protein;

[0023] 4) Using multiple rounds of mutation to perform directed mutation and post-screening of nanobodies with improved affinity

[0024] Using the MutaBind2 and mCSM-AB2 platforms to perform three rounds of prediction, selecting the amino acids with the largest change in free energy as the mutation sites, and using the QuickMutation gene site mutation kit, and the forward primer F1, the reverse primer R1; the forward primer F2, the reverse primer R2 to perform two rounds of directed mutation.

[0025] Further, the preparation method of the recombinant plasmid nanobody mutant M1, and the mutation site is R27, P28, H30, R52, G53, S55, H97, V99, L101, R102, F104, Q105, A107, Y108.

[0026] The last technical solution of the application is to provide the recombinant plasmid nanobody mutant M1 as a target for tumor diagnosis.

[0027] Compared with the existing method, the application has the following advantages and beneficial effects:

[0028] The nanobody mutant constructed in the application can improve the affinity with the antigen, improve the sensitivity and specificity of detecting HCC, and compared with the process of phage screening of a nanobody library to obtain a nanobody against a target, has the advantages of less time-consuming, easy operation, high accuracy and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The results of phage monoclonal ELISA screening of positive clones.

[0030] Figure 2 SDS-PAGE results of prokaryotic induction expression and purification of the anti-GPC3 nanobody NB-MG18.

[0031] Figure 3Results of docking of anti-GPC3 nanobody NB-MG18 and GPC3 protein are shown.

[0032] Figure 4 Results of interaction force of anti-GPC3 nanobody NB-MG18 and GPC3 protein are shown.

[0033] Figure 5 Results of SDS-PAGE of prokaryotic induced expression and purification of nanobody mutant M1 are shown.

[0034] Figure 6 Results of affinity test of nanobody mutant M1 are shown.

[0035] Figure 7 Results of specificity test of nanobody mutant M1 are shown. Specific implementation method

[0036] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0037] The raw materials used in the application and their sources are as follows:

[0038] The antibody sequence test was entrusted to Shengong Bioengineering (Shanghai) Co., Ltd.;

[0039] QuickMutation gene site mutation kit was purchased from Biyun Tian Biological Technology Co., Ltd.;

[0040] Restriction endonuclease EcoR I and Xho I, T4 ligase were all purchased from NEB company;

[0041] 2×Taq Master Mix (Dye Plus) was purchased from Novozyme Biological Technology Co., Ltd.;

[0042] DL2000 DNA marker and DL15000 DNA marker were both purchased from TaKaRa company;

[0043] Plasmid Mini Kit I D6943 plasmid extraction kit, Gel Extraction Kit D2500 agarose gel DNA recovery kit were purchased from Merck company;

[0044] The proportions not strictly limited in the application are all volume ratios;

[0045] The percentages not strictly limited in the application are all weight percentages;

[0046] The temperatures not strictly limited in the application are all room temperature.

[0047] The methods of the present application are not strictly limited, and are all performed according to conventional technical means in the art.

[0048] Example 1

[0049] S1, using phage display technology to screen anti-GPC-3 protein nanobodies, the specific steps are as follows:

[0050] (1) Using GPC-3 protein as the coated antigen for screening, dilute to 100 μL / well, add the diluted protein solution to the well plate, and place it in the 4℃ refrigerator for coating overnight;

[0051] (2) Wash the plate with PBST solution 3 times, dry it without bubbles, and then add 300 μL / well of 3% skimmed milk powder, and incubate at 37℃ for 1h;

[0052] (3) Wash the plate 3 times, dry it without bubbles, and then add 100 μL / well of the constructed phage display nanobody library (the phage display nanobody library is a full synthetic library of nanobodies with a CDR3 region of 13 amino acids length constructed in application No. CN202310089823.5), and incubate at 37℃ for 1.5h;

[0053] (4) Add 200 μL of PBST containing 0.05% Tween-20 and blow and suck 10 times, then remove the washing solution, and then add 200 μL of PBST containing 0.05% Tween-20 and blow and suck 10 times, and so on, a total of 10 times, to wash away the unbound phage, and dry with a water-absorbing paper;

[0054] (5) Add 100 μL / well of 0.1M glycine-HCl (pH=2.2) for acid elution, stand for 2min, and then add 50 μL / well of 1M Tris-HCl (pH=8.8) for neutralization;

[0055] (6) Collect the liquid in the well plate, take 2 μL of it to determine the titer, and label it as R1 Output, and transfer the remaining liquid to 2mL of freshly cultured ER2738 bacterial solution, and incubate at 37℃ for 30min;

[0056] (7) Add 6mL of preheated 37℃ LB medium and 8 μL of carbenicillin sodium (50mg / mL), and continue to culture for 2h;

[0057] (8) Add 1mL of helper phage M13K07, 91mL of LB medium and 92 μL of 50mg / mL carbenicillin sodium, and continue to culture for 2h;

[0058] (9) Add kanamycin sulfate to a final concentration of 70 μg / mL, and continue to culture overnight.

[0059] (10) The next day, centrifuge at 11,000 rpm for 20 min at 4°C, take the supernatant, add 50 mL of 2.5M PEG / NaCl solution, mix well, and stand on ice for 2 h;

[0060] (11) Centrifuge at 11,000 rpm for 30 min at 4°C, discard the supernatant, add PBS:2.5M PEG / NaCl to the resuspended precipitate at a ratio of 4:1, stand on ice for 2 h;

[0061]

[0062] (12) Centrifuge at 11,000 rpm for 30 min at 4°C, discard the supernatant, and invert the centrifuge tube on the water-absorbing paper, and invert for 3 min until the precipitate is completely dried;

[0063] (13) Add 1 mL of PBS to dissolve the precipitate, take 2 μL for titer determination, and the rest is transferred to a 1.5 mL centrifuge tube, labeled as R2 Input, and stored at -80°C. Continue to repeat three rounds of biopanning and obtain R2 Output solution, R3 Output solution, and R4 Output solution, wherein the panning conditions are set to be more complex in the second, third, and fourth rounds of panning, including the concentration of Tween-20, the number of washing times, and the concentration of GPC-3 protein coated as an antigen, as shown in Table 1.

[0064] Table 1

[0065]

[0066] S2, screening of nanobodies, the specific steps are as follows:

[0067] (1) From the R2 Output solution, R3 Output solution, and R4 Output solution, coat onto plates containing LB solid medium with CA resistance, and the next day, randomly pick single colonies when the colonies are full, inoculate into 3 mL of CA-resistant LB medium, and culture at 37°C, 220 rpm until OD 600 = 0.6;

[0068] (2) Take 2 mL of the culture and add 20 μL of helper phage M13K07, stand at 37°C for 15 min, and then place at 37°C, 220 rpm for 2 h;

[0069] (4) Add kanamycin sulfate to a final concentration of 70 μg / mL, and continue to culture overnight.

[0070] (5) The next day, centrifuge and take the supernatant for standby.

[0071] ​(6) Add 100 μL / well of GPC-3 protein solution diluted to 0.5 μg / mL on the hole plate, paste the sealing plate film, and place it in the 4°C refrigerator for coating overnight, and use skimmed milk powder as a blank control;

[0072] (7) Wash the plate with PBST for 3 times, dry with water-absorbing paper, add 350 μL / well of 3% BSA solution, and seal for 1 h at 37°C;

[0073] (8) Wash the plate for 3 times, dry with water-absorbing paper, add 100 μL / well of supernatant obtained by centrifugation in step (5), paste the sealing plate film, and incubate for 1 h at 37°C;

[0074] (9) Wash the plate for 5 times, dry with water-absorbing paper, add HRP enzyme-labeled anti-M13 secondary antibody, and incubate for 1 h at 37°C;

[0075] (10) Wash the plate for 5 times, dry with water-absorbing paper, add 100 μL / well of color developing solution (freshly prepared), place it in a 37°C water bath for incubation for 15 min, add 50 μ / L well of 2M H2SO4 to terminate the reaction; and use an enzyme-labeled instrument to measure OD 450 , and the results are shown in Figure 1 . The clone of the bacterial liquid with the highest light absorption value of positive wells / light absorption value of negative wells (Positive / Negative, P / N) is selected to send to Shanghai Biosune Biotech Co., Ltd. for sequencing, the returned sequencing results are translated into the amino acid sequence of anti-GPC-3 nanobody NB-MG18, and the amino acid sequence of anti-GPC-3 nanobody NB-MG18 is shown as SEQ ID NO. 1.

[0076] S3, nanobody expression, and the specific steps are as follows:

[0077] (1) The original strain ER2738 glycerol bacteria containing the anti-GPC-3 nanobody NB-MG18 gene is inoculated in 5 mL CA-resistant LB culture medium at a ratio of 1:1,000, and cultured overnight at 37°C and 220 rpm.

[0078] (2) The next day, the bacterial liquid of the original strain ER2738 glycerol bacteria containing the anti-GPC-3 nanobody NB-MG18 gene in step (1) is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is removed and the precipitate is retained.

[0079] (3) The plasmid containing the anti-GPC-3 nanobody NB-MG18 gene is extracted using the Plasmid Mini Kit I D6943 plasmid kit of Merck Company, and the steps are referred to the kit instruction manual.

[0080] (4) Take 1 μL of the plasmid and add it to 100 μL of E. coli TOP10F' competent cells melted on ice, and gently

[0081] Mix well; put on ice for 30 min, heat shock for 90 s in 42°C water bath, immediately cool in ice bath for 2 min; add 700 μL LB medium to centrifuge tube, shake culture at 37°C for 60 min; centrifuge at 1,000 rpm for 1 min at room temperature, discard 500 μL supernatant; resuspend the bacterial pellet, take 100 μL and add to CA-resistant LB plate, spread evenly with a disposable spreader, invert the plate, and incubate at 37°C overnight.

[0082] (5) The next day, pick a single colony with a sterile gun tip into 5 mL CA-resistant LB medium, and shake culture at 37°C overnight.

[0083] (6) The next day, take the bacterial solution and add it to 500 mL CA-resistant LB medium at a ratio of 1:100, shake culture at 37°C, 220 rpm, until the OD of the bacterial solution is 0.6, take 1 mL of the pre-induction bacterial solution, and store it at 4°C; and add IPTG at a final concentration of 0.5 mM, induce at 30°C, 220 rpm, overnight. 600

[0084] (7) The third day, centrifuge at 8,000 rpm for 10 min to collect the bacterial pellet, wash once with deionized water; freeze-thaw the bacterial pellet at -20°C, resuspend 200 mg of the pellet with 1 mL of Lysis reagent to which 10 μL of protease inhibitor, 1 μL of DTT, 2 μL of lysozyme, and 2 μL of Dnase I enzyme have been added, mix well, and shake at 280 rpm on a shaker at room temperature for 30 min.

[0085] (8) Centrifuge the lysate at 4°C, 16,000 rpm for 5 min to collect the bacterial lysate supernatant, and store it at 4°C.

[0086] (9) Take 2 mL of the mixed 50% BeyoGold TM His-tag Purification Resin, centrifuge at 4°C, 1,000 g for 10 s, and discard the 30% ethanol storage solution.

[0087] (10) Add 1 mL of PBS buffer to the gel and mix well to equilibrate the gel, centrifuge at 4°C, 1,000 g for 10 s, and retain the gel; repeat the equilibration 1-2 times by adding 1 mL of PBS buffer and retaining the gel.

[0088] (11) Mix the gel and the bacterial lysate supernatant, load into the empty column tube of the affinity chromatography column, open the lid at the bottom of the purification column, and collect about 20 μL of the flow-through.

[0089] (12) Wash the column with 2 mL of PBS buffer each time, for a total of 5 times, and collect about 20 μL of the column-washed A solution.

[0090] ​(13) Add 2 mL of washing solution to wash the column each time, for a total of 5 times, and collect about 20 μL of washing solution B that has penetrated the column.

[0091] (14) Elute the target protein with 2 mL of elution buffer each time, for a total of 5 times, and collect the elution buffer from each time into different centrifuge tubes. The collected elution buffer is the purified anti-GPC-3 nanobody NB-MG18.

[0092] (15) Perform SDS-PAGE electrophoresis detection. Rinse the electrophoresis gel preparation glass plate, short glass plate and electrophoresis sample comb with water. Place the two glass plates into the gel preparation clamp, clamp them tightly and fix them on the gel preparation frame of the electrophoresis instrument. Add deionized water and check for leakage. If there is leakage, reinstall the frame. If there is no leakage, pour out the water and use absorbent paper to absorb the water between the glass plates.

[0093] (16) Prepare a suitable 10% PAGE separating gel according to the molecular weight of the target protein. In a 10 mL centrifuge tube, add 2.08 mL of pure water, 1.67 mL of 30% Acr-Bis (29:1), and 1.25 mL of separating gel buffer, and mix thoroughly using a vortex mixer. Add 0.05 mL of 10% ammonium persulfate and 0.002 mL of N,N,N',N'-tetramethylethylenediamine (TEMED), and gently invert to mix. Slowly add the separating gel solution between the two glass plates until the solution is about 0.5 cm from the comb teeth, and then gently cover it with a 1 cm thick layer of water. Let it stand for 30 min until the separating gel solidifies and a clear interface is visible. Remove the water layer and blot away any remaining moisture with absorbent paper.

[0094] (17) Prepare a 5% SDS-PAGE stacking gel. In a 5 mL centrifuge tube, add 1.14 mL of pure water and 0.34 mL of [unclear text - possibly a typo, should be "to condense"].

[0095] Mix 30% Acr-Bis (29:1) and 0.5 mL of stacking gel buffer thoroughly using a vortex mixer; add 0.02 mL of 10% ammonium persulfate and 0.002 mL of TEMED, and gently invert the mixture to mix thoroughly, avoiding the formation of air bubbles as much as possible.

[0096] (18) Add the stacking gel solution to the separating gel until the solution slightly overflows the short glass plate, then add the sample comb.

[0097] Insert the gel into the container, being careful not to create air bubbles. Let it stand for 15 minutes until the stacking gel has polymerized. Assemble the electrophoresis apparatus, pour freshly prepared electrophoresis buffer into the inner tank, and recycled electrophoresis buffer can be used for the outer tank. Slowly pull up the sample comb and add 10 μL of the prepared sample to the wells in the predetermined order. Add the protein marker to one of the wells.

[0098] (19) Cover the electrophoresis tank with the cover, connect the power supply, and set the program to two stages: the first stage is 60V constant voltage for 30 min, and the second stage is 120V constant voltage for 60 min.

[0099] (20) After the electrophoresis is completed, carefully remove the electrophoresis gel and transfer it to a culture dish, add an appropriate amount of Coomassie brilliant blue staining solution to

[0100] completely immerse the electrophoresis gel and place it on a horizontal shaker for 30 min. After staining, recover the staining solution, rinse the electrophoresis gel with tap water, and add an appropriate amount of decolorizing solution to the horizontal shaker. Replace the decolorizing solution in small amounts and multiple times until the background color of the electrophoresis gel becomes transparent. The results are shown in Figure 2 lane 6 is the anti-GPC3 protein nanobody NB-MG18, which has a size consistent with the approximate size of a nanobody, 12-15 Kda.

[0101] S4, Homology modeling and molecular docking. The specific steps are as follows:

[0102] (1) The amino acid sequence of the anti-GPC3 nanobody NB-MG18 (SEQ ID NO. 1) is input into the Swiss-model software for homology modeling, and the top ten models are selected as candidates,

[0103] (2) The reliability and rationality of the model are comprehensively evaluated using the SAVES v6.0 website (https: / / saves.mbi.ucla.edu / ), and the homologous antibody with a similarity of 75.63% to the amino acid sequence of the anti-GPC3 nanobody NB-MG18 is selected as the best template.

[0104] (3) For the GPC3 protein model, the gene sequence of human GPC3 protein (GenBank: NM_004484.4) is obtained from GenBank, and then the gene sequence is also input into Swiss-model, and the homologous antibody with a sequence similarity of 100% is selected as the best template.

[0105] (4) Using the professional HDCOK program (http: / / hdock.phys.hust.edu.cn / ) for molecular docking, the anti-GPC3 nanobody NB-MG18 was used as the receptor, and the antigen (GPC3 protein) was used as the ligand for molecular docking to predict the structure of the antigen-antibody complex formed by the anti-GPC3 nanobody NB-MG18 and the GPC3 protein. The docking score was calculated based on the ITScorePP or ITScorePR iterative scoring function, and a more negative docking score indicates that the structural model may have greater binding and stronger interaction, so the docking score of the best docking result obtained is -250.82, and the confidence score is 0.883 (confidence score = 1.0 / [1.0 + e^0.02 * (docking score + 150)]), indicating that the complex model of the docking has a high confidence, and the model structure result is shown in Figure 3 .

[0106] (5) Then continue to use the HDCOK program to analyze the interaction mode of NB-MG18 and the GPC3 protein binding region, and the results are shown in Figure 4 , both of which are combined through hydrogen bond interaction force and many hydrophobic interaction forces, retaining the amino acids (G26, R27, P28, H30, S51, R52, G53, S55, H97, V99, G100, L101, R102, F104, Q105, A107, Y108) that produce the binding effect.

[0107] S5, analyze the key amino acids of the docking. The specific steps are as follows:

[0108] (1) Using the bioinformatics online website InterProSurf to analyze the key amino acids (R27, P28, H30, R52, G53, S55, H97, V99, G100, L101, R102, F104, Q105, A107, Y108) of the nanobody that produce the interaction interface of NB-MG18 and GPC3.

[0109] (2) Further using Pymol to analyze the key amino acids (R27, P28, H30, R52, G53, S55, H97, V99, L101, R102, F104, Q105, A107, Y108) of the nanobody that produce the interaction interface of NB-MG18 and GPC3.

[0110] (3) Cross-select the amino acids analyzed by HDCOK in S5 example with the amino acids analyzed by InterProSurf and PyMOL, remove the amino acid sites without overlap (G26, S51, G100), and obtain the final mutation sites: R27, P28, H30, R52, G53, S55, H97, V99, L101, R102, F104, Q105, A107, Y108. The results are shown in Table 2.

[0111] Table 2

[0112]

[0113] S5, after analyzing multiple rounds of mutations, the nanobodies with improved affinity were screened after directed mutation, and the specific steps were as follows:

[0114] (1) The first round of amino acid mutation was performed using mCSM-AB2 and MutaBind2. Considering the specificity of cysteine and alanine, 11 interface residues (R27, P28, H30, R52, G53, S55, H97, V99, L101, R102, F104, Q105, A107, Y108) were mutated to 18 other natural amino acids (except cysteine and alanine) in the first round of calculation, and 198 single-point mutations were obtained.

[0115] (2) In the second round of calculation, the multiple mutation module of MutaBind2 was used for double mutation prediction, and the results are shown in Table 3.

[0116] Table 3

[0117]

[0118] (3) In the third round of calculation, the multiple mutation module of MutaBind2 was used for triple mutation prediction, and the results are shown in Table 4.

[0119] Table 4

[0120]

[0121] All these mutants were selected according to the standard of ΔΔG<-2, and the most negative ΔΔG value was selected from the parent nanobody (NB-MG18). As can be seen from Table 4, the most negative value is the ΔΔG of triple mutant H30N&P28F&L101Q =-3.58 (SEQ ID NO. 2), and the triple mutant H30N&P28F&L101Q is named M1, and then subsequent expression is performed.

[0122] (5) Design forward and reverse primers F1, R1, F2, R2, and the primer sequences are as follows:

[0123] Forward primer F1: 5'-CTGTACAGCCTCTGGTAGGTTCTCTAATACTGGTATGAT-3'

[0124] (SEQ ID NO. 3);

[0125] Reverse primer R1: 5'-GACATGTCGGAGACCATCCAAGAGATTATGACCATACTA-3', (SEQ ID NO. 4);

[0126] Forward primer F2: 5'-TGCACATGGCGTGGGACAACGGGCCTT-3' (SEQ ID NO. 5);

[0127] Reverse primer R2: 5'-ACGTGTACCGCACCCTGTTGCCCGGAA-3' (SEQ ID NO. 6).

[0128] The amino acid position HIS30 of the anti-GPC3 nanobody NB-MG18 is mutated to ASN30, and PRO28 is mutated to PHE28 by using the QuickMutatio gene site-directed mutagenesis kit and the designed primers (F1, R1); the amino acid position LEU101 of the anti-GPC3 nanobody NB-MG18 is mutated to GLN101 by using the QuickMutatio gene site-directed mutagenesis kit and the designed primers (F2, R2),

[0129] The final amino acid sequence is SEQ ID NO. 2, and the specific steps refer to the QuickMutatio gene site-directed mutagenesis kit instructions.

[0130] (4) The PCT product and the pComb 3xss plasmid are respectively digested with restriction endonuclease EcoR I and Xho I at 37°C for 16 h, and the digested target fragments and pComb 3xss vectors are recovered by the Gel Extraction Kit D2500 kit, and the steps refer to the Gel Extraction Kit D2500 kit instructions.

[0131] (5) T4 DNA ligase is used for ligation at 16°C overnight.

[0132] (6) 3 μl of the ligation product is mixed with 100 μl of TOF10F` competent cells, and then ice bathed for 25 minutes, followed by heat shock at 42° for 60 s and then immediately ice bathed for 3 minutes, and then 600 μl of culture medium is added and cultured in a 37°, 220 rpm incubator for 1 h.

[0133] (7) In the LB plate containing 100 μg / mL carbenicillin, 12 h, pick the single colony with good morphology to carry out bacterial liquid PCR identification, and send the positive single colony liquid to sequencing identification. The correct identification result of the recombinant plasmid nanobody mutant M1 (SEQ ID NO. 2) is stored.

[0134] (8) The mutant nanobody M1 is expressed, and the operation steps are the same as those in Example S3. The results are shown in Figure 5 , and the antibody size is consistent with the 12-15 KDa size of the nanobody.

[0135] In order to verify the effect of the GPC3 nanobody M1 provided in the application, the affinity determination of the mutant anti-GPC3 nanobody M1 and the GPC3 protein and the specificity determination of the mutant anti-GPC3 nanobody M1 and the GPC3 protein are given below, and the experimental results are given below.

[0136] I. Affinity determination of mutant anti-GPC3 nanobody M1 and GPC3 protein

[0137] The specific steps are as follows:

[0138] (1) Coat GPC3 protein as antigen in microplate and place at 4°C overnight.

[0139] (2) After washing 3 times with PBST containing 0.05% Tween-20, block with 5% BSA in PBST at 37°C for 1 hour.

[0140] (3) Wash the microplate, then add 100 μl of mutant nanobody M1 per well, incubate at 37°C for 1 hour, and use PBS and NB-MG18 as controls.

[0141] (4) Wash the microplate, and incubate with HRP-labeled secondary antibody at 37°C for 1 hour.

[0142] (5) Wash the microplate, add TMB two-component substrate solution to observe the reaction results. After incubation at 37°C for 15 min, stop the reaction with 50 μl of 2M H2SO4, and measure the absorbance value of each well at 450 nm with an enzyme marker. The results are shown in Figure 6 , and it can be seen from Figure 6 that the binding ability of the mutant nanobody M1 protein to the GPC3 protein is better than that of the PBS solution, and the OD value is 1.732. Compared with the NB-MG18 nanobody protein, the binding ability of the mutant nanobody M1 is significantly improved.

[0143] II. Specificity determination of mutant anti-GPC3 nanobody M1 and GPC3 protein

[0144] The specific steps are as follows:

[0145] (1) After the GPC3 protein is treated, SDS-PAGE protein electrophoresis is performed, and the operation steps are the same as those in Example S4. The separating gel containing the GPC3 protein, two transfer sponges, and two transfer filter papers are soaked in the transfer buffer; one 5x8 cm PVDF membrane is first soaked in 100% methanol until it becomes transparent (about 1-2 min), and then placed in the transfer buffer for equilibration. The transfer clamp is placed in the transfer slot according to the principle of “black to black, red to white”, and the pre-cooled transfer buffer is added.

[0146] (2) The transfer device is placed in an ice water bath, the power is turned on, and the transfer is performed at a constant current of 300 mA for 60-70 min, at which time the GPC3 protein is transferred from the separating gel to the PVDF membrane. After the transfer is completed, the PVDF membrane is rinsed with TBST buffer.

[0147] (3) The PVDF membrane is soaked in a WB incubation box containing 5% skim milk powder TBST buffer, and blocked for 2 h on a horizontal shaker at room temperature at 140 rpm;

[0148] (4) After blocking, the PVDF membrane is rinsed with TBST 3 times for 5 min each time; a solution of mutant nanobody M1 diluted in 5% skim milk powder TBST buffer is added, and incubated at 4°C overnight;

[0149] (5) The next day, the PVDF membrane is washed with TBST 3 times for 10 min each time; a HRP enzyme-labeled goat anti-HA secondary antibody diluted in 5% skim milk powder TBST is added, and incubated at 140 rpm and room temperature for 2 h;

[0150] (6) After incubation, the PVDF membrane is washed with TBST 3 times for 10 min each time, the water on the membrane is removed with filter paper, the PVDF membrane is placed on a clean plastic culture dish, the developing solution is evenly applied to the PVDF membrane, and the excess developing solution is removed; the ChemiDoc XRS+ imaging system of Biorad Company is used for development, and the results are shown in Figure 7 It can be seen from Figure 7 that lane No. 2 has only a single band with a size of about 70 Kda, which is consistent with the size of GPC3 protein, and there is no other band, indicating that the mutant nanobody M1 has good specific binding ability with GPC3.

[0151] The results show that the mutant nanobody M1 not only has a significant improvement in affinity compared with the anti-GPC-3 nanobody NB-MG18, but also can be well combined with the GPC3 protein, which is conducive to the development of HCC detection reagents.

[0152] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A recombinant plasmid nanoantibody mutant M1, characterized in that, The amino acid sequence of the recombinant plasmid nanobody mutant M1 is shown in SEQ ID NO. 2; and it can specifically bind to GPC3 protein.

2. The method for preparing the recombinant plasmid nanoantibody mutant M1 according to claim 1, characterized in that, The recombinant plasmid nanobody mutant M1 was obtained by two rounds of mutation of the anti-GPC3 nanobody NB-MG18; Using a gene site-directed mutagenesis kit and primer set, the amino acid positions of the anti-GPC3 nanobody NB-MG18 were mutated from HIS30 to ASN30, PRO28 to PHE28, and LEU101 to GLN101, resulting in the recombinant plasmid nanobody mutant M1 with the amino acid sequence shown in SEQ ID NO.

2. The amino acid sequence of the anti-GPC3 nanobody NB-MG18 is shown in SEQ ID NO. 1; When HIS30 is mutated to ASN30 and PRO28 is mutated to PHE28, the nucleotide sequence of the forward primer F1 is shown in SEQ ID NO. 3; the nucleotide sequence of the reverse primer R1 is shown in SEQ ID NO.

4. When LEU101 is mutated to GLN101, the nucleotide sequence of the forward primer F2 is shown in SEQ ID NO. 5; the nucleotide sequence of the reverse primer R1 is shown in SEQ ID NO.

6.

3. The method for preparing the recombinant plasmid nanoantibody mutant M1 according to claim 2, characterized in that, The site-directed mutagenesis kit used was the QuickMutatio site-directed mutagenesis kit.

4. The method for preparing the recombinant plasmid nanoantibody mutant M1 according to claim 3, characterized in that, The steps are as follows: 1) Homologous modeling Using the anti-GPC3 nanobody as the initial sequence, the three-dimensional structure of the nanobody was constructed using homology modeling technology, and after evaluation by SAVES, a homologous antibody with 100% sequence similarity was selected as the template. 2) Molecular docking Protein-protein docking was performed using the HDCOK program, with the anti-GPC3 nanobody NB-MG18 as the acceptor and GPC3 protein as the ligand, to predict the structure of the antigen-antibody complex formed by the anti-GPC3 nanobody NB-MG18 and GPC3 protein. 3) Analyze the key amino acids for docking. The online websites InterProSurf and PyMOL were used to analyze and predict the amino acid residues of nanobodies located at the interface with the GPC3 protein. 4) Screening for nanobodies with enhanced affinity after targeted mutation using multiple rounds of mutation. Three rounds of prediction were performed using the MutaBind2 and mCSM-AB2 platforms. The amino acid with the most changes in free energy was selected as the mutation site. Two rounds of targeted mutagenesis were performed using the QuickMutatio gene site-directed mutagenesis kit, forward primer F1, reverse primer R1, forward primer F2, and reverse primer R2.

5. The method for preparing the recombinant plasmid nanoantibody mutant M1 according to claim 4, characterized in that, The mutation sites in step 4) are R27, P28, H30, R52, G53, S55, H97, V99, L101, R102, F104, Q105, A107, Y108.

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