Nano-antibodies targeting tissue factor and preparation methods and applications thereof

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a nano antibody targeting tissue factor and a preparation method and application thereof. The present invention obtains two nano antibodies with high specificity and high affinity by screening from a constructed nano antibody library, which can specifically bind to tissue factor protein and can be used for detecting tissue factor on the surface of tumor cells and detecting the expression of tumor cell tissue factor by Western Blot method, thereby providing a method for studying the function of tissue factor.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a nano antibody targeting tissue factor and a preparation method and application thereof. Background Art

[0002] In recent years, with the in-depth understanding of the role of tissue factor in the formation and development of tumors, tumor therapy targeting tissue factor (TF) has become a hot topic of research. At present, anti-tumor inhibitors that directly target tissue factor mainly include TF immunoconjugates and TF monoclonal antibodies. TF immunoconjugates use FVII as a specific binding region to bind to TF specifically expressed on the surface of tumor cells, coupled or combined with Fc effector regions, tumor drugs, chemotherapy drugs, etc., to target and kill tumor cells to achieve the purpose of tumor treatment. In addition, tissue factor monoclonal antibodies have also shown good anti-cancer effects. CV. Ngo et al. used CNTO-859 antibodies to target and inhibit tissue factor in mouse breast cancer [Combination antibody therapy targeting both xenograft-and host-derived tissue factor leads to potent tumor inhibition. Cancer Research 05 / 01 / 2005]. Versteeg HH et al. showed that the antibody Mab-10H10 can block the binding of TF to integrin β1 and inhibit tumors [Henri HVersteeg, FlorenceSchaffner, Marjolein Kerver, Helle H Petersen, Wolfram Ruf. Inhibition of tissue factor signaling suppresses tumor growth. 10.1182 / blood-2007-07-101048]. Antibodies targeting tissue factor have shown significant anti-cancer activity, but there are few studies on tissue factor nanoantibodies. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a nano antibody targeting tissue factor and its preparation method and application. The tissue factor nano antibody provided by the present invention can specifically bind to tissue factor protein; it can be used to detect tissue factor on the surface of tumor cells and to detect the expression of tissue factor in tumor cells by Western Blot method, providing a method for studying the function of tissue factor.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a nanobody targeting tissue factor, wherein the nanobody comprises: a nanobody 1 having an amino acid sequence as shown in SEQ ID NO.1 and / or a nanobody 2 having an amino acid sequence as shown in SEQ ID NO.2.

[0006] The present invention also provides a coding sequence of the nanobody described in the above technical solution, wherein the coding sequence comprises: a coding sequence 1 such as a nucleotide sequence as shown in SEQ ID NO.3 and / or a coding sequence 2 such as a nucleotide sequence as shown in SEQ ID NO.4.

[0007] The present invention also provides an expression vector containing the coding sequence described in the above technical solution.

[0008] Preferably, the basic vector of the expression vector includes pET-28a vector.

[0009] The present invention also provides a host bacterium containing the expression vector described in the above technical solution.

[0010] Preferably, the host bacteria includes Escherichia coli BL21 (DE3).

[0011] The present invention also provides a method for preparing the nanobody described in the above technical solution, comprising the following steps:

[0012] Using tissue factor protein as an antigen to immunize alpacas to obtain immunized alpaca serum;

[0013] RNA from lymphocytes in immunized alpaca serum was extracted and reverse transcribed to obtain cDNA;

[0014] Using the cDNA as a template, using primer pair 1 to perform a first round of PCR amplification to obtain an amplified product; using the amplified product as a template, using primer pair 2, primer pair 3 and primer pair 4 to perform a second round of PCR amplification to obtain three amplified fragments;

[0015] The three amplified fragments are respectively connected to the phage vector to construct a nanobody library;

[0016] Screening a nanobody with relatively high specificity and affinity from the nanobody library to obtain the nanobody according to claim 1;

[0017] The primer pair 1 consists of F1 and R1; the primer pair 2 consists of F2 and R2; the primer pair 3 consists of F2 and R3; the primer pair 4 consists of F4 and R4;

[0018] The nucleotide sequence of F1 is shown in SEQ ID NO.5; the nucleotide sequence of R1 is shown in SEQ ID NO.6;

[0019] The nucleotide sequence of F2 is shown in SEQ ID NO.7; the nucleotide sequence of R2 is shown in SEQ ID NO.8;

[0020] The nucleotide sequence of R3 is shown in SEQ ID NO.9;

[0021] The nucleotide sequence of F4 is shown in SEQ ID NO.10; the nucleotide sequence of R4 is shown in SEQ ID NO.11.

[0022] Preferably, the screening method comprises solid phase panning.

[0023] The present invention also provides the use 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 host bacteria described in the above technical solution, or the nanobody prepared by the preparation method described in the above technical solution in the preparation of a reagent for detecting tissue factor expression.

[0024] Preferably, the sample to be detected includes tumor tissue or tumor cells.

[0025] Beneficial effects:

[0026] The present invention provides a nano antibody targeting tissue factor, the nano antibody comprising: a nano antibody 1 having an amino acid sequence as shown in SEQ ID NO.1 and / or a nano antibody 2 having an amino acid sequence as shown in SEQ ID NO.2. The present invention screens and obtains two nano antibodies with high specificity and high affinity from a constructed nano antibody library, which can specifically bind to tissue factor protein, can be used for the detection of tissue factor on the surface of tumor cells and the detection of the expression of tissue factor in tumor cells by Western Blot method, and provides a method for the study of tissue factor function. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 This is the electropherogram of the first round of PCR amplification;

[0029] Figure 2 This is the electropherogram of the second round of PCR amplification;

[0030] Figure 3 The results of the purification of nanobody and Fc fusion expression; the lanes from left to right are nanobody 1, nanobody 2 and Maker; Maker from top to bottom are 116kDa, 66kDa, 45kDa, 35kDa, 25kDa, 18kDa, 12kDa;

[0031] Figure 4 This is the affinity test result of Nanobody 1;

[0032] Figure 5 This is the affinity test result of Nanobody 2;

[0033] Figure 6 Western-Blot detection of the expression of tissue factor on the surface of tumor cells; the lanes from left to right are Nanobody 1, Nanobody 2 and Maker; Maker is 35kDa;

[0034] Figure 7 The results of detecting the expression of tissue factor on the cell surface by fluorescently labeled nanoantibody 1; from top to bottom are the results of tumor cell surface fluorescent labeling, DAPI cell nucleus and Merge;

[0035] Figure 8 The results of detecting the expression of cell surface tissue factor by fluorescently labeled nanoantibody 2; from top to bottom are the tumor cell surface fluorescent labeling results, DAPI cell nucleus and Merge. DETAILED DESCRIPTION

[0036] The present invention provides a nanobody targeting tissue factor, the nanobody comprising: a nanobody 1 with an amino acid sequence as shown in SEQ ID NO.1 and / or a nanobody 2 with an amino acid sequence as shown in SEQ ID NO.2, the specific amino acid sequences are as follows:

[0037] SEQ ID NO.1: QLVESGGGLVQPGGSLRLSCVASGFTFDSHDMAWYRQA PGKKCELVAATNSDGSSTYYAHSVKGRFTVSRDNAENTAWLQMNDLKSEDTA VYYCNGDTTRVGPSSSACAGGVSPEYWGQGTQVTVSSGR;

[0038] SEQ ID NO. 2: QLAESGGGLVQAGGSLRLSCVASGRTTSSYYVMGWFRQD SGKEREFVATIRWRGDSYYADSVKGRFTISRDNAKNTTYLQMNSLKPEDTAV YYCAVATYSGTLYLTRGFEYNYWGQGTQVTVSSGR.

[0039] The nano antibody provided by the present invention is fused with the Fc fragment for expression and purification. The molecular weight after expression is 40 kD, the purity is more than 95%, and the affinity between the nano antibody and the tissue factor protein is KD=10 -9M and above, it can specifically recognize tissue factor protein on the cell surface and has the advantages of high specificity and high affinity.

[0040] The present invention also provides a coding sequence of the nanobody described in the above technical solution, wherein the coding sequence comprises: a nucleotide sequence such as a coding sequence 1 as shown in SEQ ID NO.3 and / or a nucleotide sequence such as a coding sequence 2 as shown in SEQ ID NO.4, and the specific nucleotide sequence is as follows:

[0041] SEQ ID NO.3: 5'-CAGCTTGTCGAAAGCGGCGGAGGCCTAGTGCAGCC TGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCGATAGTCATGACATGGCCTGGTACCGTCAGGCTCCAGGGAAGAAATGCGAGTTGGTCGCAGCGACTAACAGTGATGGGAGTAGTACGTATTATGCACACTCCGTTAAGGGCCGATTCACCGTCTCCAGAGAC AATGCCGAGAACACGGCGTGGCTCCAAATGAACGACCTGAAATCTGAGGACACGGCCGTGTATTACTGTAATGGAGATACGACCCGTGTGGGCCCAAGTTCTTCGGCCTGTGCAGGCGGTGTGTCGCCCGAGTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGGCCGG-3';

[0042] SEQ ID NO.4: 5'-CAGCTTGCCGAGTCTGGGGGAGGATTGGTGCAGGC TGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCTCCAGTTACTATGTCATGGGCTGGTTCCGCCAGGATTCAGGGAAGGAGCGTGAGTTTGTAGCAACTATTAGATGGAGAGGTGATAGTTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAA CGCCAAGAACACAACGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGCCGTTTATTACTGCGCAGTCGCAACCTATAGTGGTACTTTGTATCTCACGCGGGGTTTTGAGTATAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGGCCGGCCTGGCCAGGC-3'.

[0043] The coding sequence 1 described in the present invention is a sequence encoding nanobody 1, and the coding sequence 2 is a sequence encoding nanobody 2.

[0044] The present invention also provides an expression vector containing the coding sequence described in the above technical solution.

[0045] In the present invention, the basic vector of the expression vector preferably includes a pET-28a vector.

[0046] The present invention also provides a host bacterium containing the expression vector described in the above technical solution.

[0047] In the present invention, the host bacteria preferably includes Escherichia coli BL21 (DE3).

[0048] The present invention also provides a method for preparing the nanobody described in the above technical solution, comprising the following steps:

[0049] Using tissue factor protein as an antigen to immunize alpacas to obtain immunized alpaca serum;

[0050] RNA from lymphocytes in immunized alpaca serum was extracted and reverse transcribed to obtain cDNA;

[0051] Using the cDNA as a template, using primer pair 1 to perform a first round of PCR amplification to obtain an amplified product; using the amplified product as a template, using primer pair 2, primer pair 3 and primer pair 4 to perform a second round of PCR amplification to obtain three amplified fragments;

[0052] The three amplified fragments are respectively connected to the phage vector to construct a nanobody library;

[0053] Screening nanobodies with relatively high specificity and affinity from the nanobody library to obtain the nanobodies described in the above technical solution;

[0054] The primer pair 1 consists of F1 and R1; the primer pair 2 consists of F2 and R2; the primer pair 3 consists of F2 and R3; the primer pair 4 consists of F4 and R4;

[0055] The nucleotide sequence of F1 is shown in SEQ ID NO.5, which is as follows: 5'-GTCCTGGCTGCTCTTCTACAAGG-3';

[0056] The nucleotide sequence of R1 is shown in SEQ ID NO.6, which is as follows: 5'-GGTACGTGCTGTTGAACTGTTCC-3';

[0057] The nucleotide sequence of F2 is shown in SEQ ID NO.7, which is as follows: 5'-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3';

[0058] The nucleotide sequence of R2 is shown in SEQ ID NO.8, which is as follows: 5'-GGCCAGGCCGGCCTGAGGAGACGGTGACCTGGGT-3';

[0059] The nucleotide sequence of R3 is shown in SEQ ID NO.9, which is as follows: 5'-GGCCAGGCCGGCCACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3';

[0060] The nucleotide sequence of F4 is shown in SEQ ID NO.10, which is as follows: 5'-GGCCCAGGCGGCCGAGGTGCAGCTGSWGSAKTCKG-3';

[0061] The nucleotide sequence of R4 is shown in SEQ ID NO.11, which is as follows: 5'-GGCCAGGCCGGCCTGAGGAGACGGTGATGACCWGGGTC-3'; wherein S, W, and K are degenerate bases, S is C / G; W is A / T; and K is G / T.

[0062] In the present invention, the phage vector preferably includes a pComb3xss vector; and the screening method preferably includes solid phase panning.

[0063] The nanoantibody TGF-β1-VHH was expressed using phage display technology; the nanoantibody with high binding affinity to the antigen was screened out using bio-screening technology

[0064] The library capacity and diversity of the nanoantibody library are the key to selecting high-specificity and high-affinity nanoantibodies. The present invention uses primer pair 2, primer pair 3 and primer pair 4 to perform PCR amplification to obtain three amplified fragments, and connects the three amplified fragments to the phage vector to construct a nanoantibody library with a capacity of 2.8×10 10 PFU, antibody fragment diversity greater than 95% of the nanoantibody library. Phage display technology is used to display nanoantibody proteins on the surface of phages. The displayed peptides or proteins can maintain relatively independent spatial structure and activity, which is conducive to the recognition and binding of target molecules. Through solid phase panning, nanoantibodies with high specificity and high affinity are obtained.

[0065] The present invention also provides the use 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 host bacteria described in the above technical solution, or the nanobody prepared by the preparation method described in the above technical solution in the preparation of a reagent for detecting tissue factor expression.

[0066] In the present invention, the sample to be detected preferably includes tumor tissue or tumor cells.

[0067] The nano antibody provided by the present invention can specifically bind to tissue factor protein; it can be used to detect tissue factor on the surface of tumor cells and to detect the expression of tissue factor in tumor cells by Western Blot, providing a method for studying the function of tissue factor.

[0068] To further illustrate the present invention, the nanoantibodies targeting tissue factor and their preparation methods and applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0069] Example 1

[0070] 1. Alpaca immunization with tissue factor protein

[0071] Alpacas from Qifeng Goat Camel Breeding Base in Yangqu County, Taiyuan City were selected. Healthy and strong alpacas with good mental state and moderate body shape were selected. The selected alpacas had bright fur color, no symptoms of injury or discomfort, and no immunization records. After recording the ear numbers of the selected alpacas, the immunization experiment was started. Each time, the alpacas were injected on both sides near the lymph nodes in the neck, with 2 injections on each side. The antigen was tissue factor protein. The amount of antigen for the first immunization was 600 micrograms, and the adjuvant was Freund's complete adjuvant (sigma). The volume ratio of antigen to adjuvant was 1:1; the amount of antigen for the second and third immunizations was 600 micrograms, and the adjuvant was Freund's incomplete adjuvant (sigma); the amount of antigen for the fourth to sixth immunizations was 1 milligram, and the adjuvant was Freund's incomplete adjuvant; after the third immunization, 1 mL of blood was drawn each time for detecting serum titer; the amino acid sequence of the antigen is as shown in SEQ ID As shown in NO.13: SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREIFYIIGAVVFVVIILVIILAISLH; the construction method of the antigen refers to Chinese patent CN200510012414.7.

[0072] The indirect ELISA method was used to determine the titer of alpaca serum after immunization. The secondary antibody was anti-alpaca IgG-HRP labeled antibody. The titer of alpaca serum after the sixth immunization was 1:4000000.

[0073] 2. Construction of tissue factor nanoantibody phage library

[0074] Draw 50mL of alpaca serum after the sixth immunization and divide it into 5 anticoagulant tubes, 10mL in each tube, and centrifuge directly in the anticoagulant tubes at room temperature, 2000rpm, for 10 minutes; the supernatant is serum, which can be frozen for use, and be careful not to damage the white film (cell layer); add PBS buffer (containing 2% FBS) in an amount equal to the remaining volume and blow it gently; prepare another 15mL centrifuge tube, add 5mL Ficol separation solution, and gently add 10mL of cell suspension along the wall to keep the layers intact. Centrifuge at 2000rpm for 30 minutes, set the centrifuge speed to 0; slowly wash out the white membrane (using the pipette in the Lonza electroporation), add the white membrane to a new 15mL centrifuge tube, add 5mL PBS buffer (containing 2% FBS) to the centrifuge tube, centrifuge at 2500rpm for 10 minutes; discard the supernatant, add 5mL PBS buffer (containing 2% FBS), wash once, count, and centrifuge at 2500rpm for 10 minutes; discard the supernatant, add cell lysis buffer according to the cell count, 10 per tube 7 cells.

[0075] Lymphocyte RNA was extracted by TRIZOL lysis method, reverse transcribed into cDNA, and the nanobody gene fragment was amplified by nested PCR. The nucleotide sequence of the upstream primer of the first round of PCR is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6; the first round of PCR reaction program is: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1 minute, 30 cycles; 72℃, 10min. A 0.7kb gene fragment ( Figure 1 ); the fragment was recovered from the gel and used as a template for the second round of amplification. The second round of PCR amplification used three different pairs of primers (primer pair 2, primer pair 3 and primer pair 4) for amplification respectively; the second round of PCR reaction program was 95°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 57°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles; 72°C, 10 minutes; the information of the three different primer pairs is as follows:

[0076] Primer pair 2:

[0077] VHH-BACK-F(F2): 5'-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3' (SEQ ID NO.7);

[0078] PMCF-R(R2): 5'-GGCCAGGCCGGCCTGAGGAGACGGTGACCTGGGT-3' (SEQ ID NO. 8);

[0079] Primer pair 3:

[0080] VHH-BACK-F(F3): 5'-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3' (SEQ ID NO.7);

[0081] VHH-For-R(R3): 5'-GGCCAGGCCGGCCACTAGTGCGGCCGCTGGAG ACGGTGACCTGGGT-3' (SEQ ID NO.9);

[0082] Primer pair 4:

[0083] VHHF (F4): 5'-GGCCCAGGCGGCCGAGGTGCAGCTGSWGSAKTCKG-3' (SEQ ID NO. 10);

[0084] VHHR (R4): 5'-GGCCAGGCCGGCCTGAGGAGACGGTGATGACCWGG GTC-3' (SEQ ID NO. 11);

[0085] Where S is C / G; W is A / T; K is G / T;

[0086] A 0.4 kb gene fragment was obtained by amplification ( Figure 2), the three different fragments amplified in the second round were mixed and then digested with sfiI enzyme, the digestion reaction system was: 25μL PCR fragment, 10μL sfiI enzyme, 10μL10×buffer and 55μL water; the digestion condition was 50℃ overnight; after digestion, the 0.4kb fragment was recovered; the recovered digested fragment was connected with the phage vector pComb3xss, the connection system and conditions were: 18μL digested fragment, 14μL10×buffer, 9μL T4 ligase, 23μLpComb3xss and 86μL water, and the connection was carried out at 16℃ overnight; after connection, it was divided into 10 parts and electroporated into 10 XL-Blue competent cells respectively, and 1mL SOC medium was added to each after electroporation, the electroporation cup was washed with SOC medium, and then the combination was cultured at 37 degrees for 1h, 10μL was added to 90μL SOC medium, and 10μL was added to 90μL after mixing. In SOC medium, apply ampicillin plate, then add 5mL SOC medium (total volume 15mL), add 3μL ampicillin, 30μL tetracycline, and culture at 37 degrees and 250rpm for 1 hour. After 1 hour, add 185mL SB medium, add 93μL ampicillin, 370μL tetracycline, total volume 200mL, and then add 2mL helper phage VCSM13, culture at 37 degrees for 2 hours, then add 280μL kanamycin to terminate, and culture overnight; centrifuge the overnight bacterial solution at 7000rpm, 4 degrees for 15 minutes to obtain the supernatant; at the same time, use a new centrifuge bucket to weigh 4g PEG 8000 and 3g sodium chloride based on 100mL of overnight bacterial solution;

[0087] Library collection: the supernatant was mixed with the weighed PEG8000 and sodium chloride, and ice-bathed for 30 minutes; centrifuged at 9500rpm, 4 degrees for 20 minutes, and the precipitate was retained and resuspended with 2mL PBS (PBS filtered with 0.22μm); centrifuged at 14000rpm for 5 minutes; the supernatant was filtered through a 0.22mm filter into a new 2mL centrifuge tube to obtain the phage library; the library capacity was tested on a plate, and the test library capacity was 2.8×10 10 PFU. 38 sequences were sequenced with 0 duplicates and 100% sequence diversity.

[0088] 3. Screening and expression of tissue factor nanoantibodies. The specific steps are as follows:

[0089] (1) Amplification of phage library: Add 50 mL SB medium, 100 μL tetracycline and 5 μL XL-Blue bacteria to a 250 mL Erlenmeyer flask and culture at 37°C in a shaking incubator until OD 6001; transfer to a 500mL Erlenmeyer flask, add 10μL of the phage library constructed in step 2, and infect at room temperature for 15 minutes; add 50μL of ampicillin and shake at 37 degrees for 1 hour; add 148mL SB medium, 2mL helper phage (VCSM13), 300μL of tetracycline and 75μL of ampicillin, and culture at 37℃ for 2 hours; add 280μL of kanamycin and incubate at 300rpm overnight.

[0090] (2) The method of storage is the same as step 2.

[0091] (3) Enrichment: 96-well ELISA plates were coated with tissue factor protein (amino acid sequence as shown in SEQ ID NO.13, coating concentration was 1 μg / well), incubated at 4°C overnight, and blocked with 3% BSA for 1 h; 100 μL of phage library was added and incubated at 37°C for 2 h; de-library: wells were washed with 200 μL of PBS solution containing 0.5% Tween 20, and the first round of washing was repeated five times. Wash 10 times in the second round, 15 times in the third and fourth rounds, each time gently aspirate the wash solution with a pipette, repeatedly blow the wells 10 times, and then discard it; 50μL of 10mg / ml trypsin, digest at 37℃ for 30 minutes; transfer all digestion products into 2mL XL-Blue, infect at room temperature for 15 minutes; add 6mL SB medium and 12μL tetracycline, add ampicillin at a final concentration of 50μg / mL, and shake at 37℃; add 91mL SB medium, 1mL helper phage, 182μL tetracycline and 46μL ampicillin, incubate at 37℃ for 2 hours, add 140μL kanamycin, and incubate at 37℃ overnight.

[0092] (4) Collect the library. Centrifuge the overnight bacteria at 7000 rpm and 4°C for 15 min. At the same time, weigh 4 g PEG8000 and 3 g sodium chloride in a new centrifuge bucket (the amount for 100 mL of bacterial solution should be doubled if it is 200 mL). Use the supernatant obtained from the centrifugation to dissolve the PEG8000 and sodium chloride. Place in an ice bath for 30 min. Centrifuge at 9500 rpm and 4°C for 20 min. Keep the precipitate. Resuspend the precipitate in 2 mL of TBS containing 1% BSA (filtered through a 0.22 mm filter). Centrifuge at 14000 rpm and 4°C for 5 min. Pass the supernatant through a 0.22 mm filter into a new 2 mL EP tube. Dilute the collected phage library by 10 -7 , 10 -8 , 10 -9 , take 1 μL to infect 50 μL XL-Blue, infect at room temperature for 15 minutes, apply ampicillin plates, culture overnight, and obtain monoclonal colonies.

[0093] (5) Screening: Add 800 μL of SB medium containing 1.6 μL of tetracycline and 0.8 μL of ampicillin to each well of a 96-well plate, pick a single clone, and place it into the deep-well plate together with the pipette tip, and shake at 37°C, 300 rpm for 6 h; Bacteria preservation: spot the agar plate with the pipette tip (mark the plate in advance); add 100 μL of SB medium containing 0.2 μL of tetracycline and 1 μL of ampicillin to each well, add 20 μL of helper phage, and shake at 37°C, 300 rpm for 2 h; add 2.8 μL of kanamycin to each well, and shake overnight.

[0094] ELISA test, positive clones were sent for sequencing. Sequence alignment was performed by NCBI and it was determined to be an alpaca nanobody sequence, the amino acid sequence of the nanobody is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0095] The alpaca nanobody sequence is fused with the human antibody Fc sequence for expression; the amino acid sequence of the Fc sequence is shown in SEQ ID NO.12, and is specifically as follows: ASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVRVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0096] The linker and Fc sequences of the two screened nanobodies were codon optimized and synthesized at BGI. The linker was (G4S)3 amino acids, and the optimized base sequences were as follows:

[0097] Nanobody 1 is shown as SEQ ID NO.14: CAACTGGTGGAATCTGGTGGTGGT CTGGTTCAACCTGGTGGCAGCCTGCGCCTGTCCTGCGTCGCGTCTGGCTTTACCTTTGATTCTCACGACATGGCGTGGTATCGTCAGGCGCCAGGTAAAAAATGCGAACTGGTGGCTGCAACCAACTCTGATGGTTCCTCTACCTACTACGCGCACTCCGTAAAAGGTCGTTTCACCGTATCCCGTGACAACGCTGAAAACACGGCGTGGCTGCAGATGAACGACCTGAAATCTGAAGACACTGCCGTCTACTATTGCAACGGTGACACTACCCGTGTTGGTCCATCTTCTTCTGCTTGCGCTGGTGGTGTATCTCCGGAGTATTGGGGTCAGGGTACCCAGGTAACTGTGTCTAGCGGCCGT;

[0098] Nanobody 2 is shown as SEQ ID NO.15: CAACTGGCGGAATCTGGTGGTGGT CTGGTTCAAGCTGGCGGCTCCCTGCGTCTGTCTTGTGTTGCATCCGGTCGTACTTCTTCCTACTACGTGATGGGCTGGTTCCGTCAGGACAGCGGCAAAGAACGTGAGTTCGTTGCTACCATTCGTTGGCGTGGTGACAGCTATTACGCAGACTCCGTTAAAGGTCGTTTCACGATCAGCCGCGACAACGCTAAAAACACCACCTACCTGCAGATGAACTCCCTGAAACCGGAGGATACCGCGGTGTATTACTGCGCAGTGGCCACCTACTCCGGTACTCTGTATCTGACCCGCGGCTTTGAGTACAACTATTGGGGCCAGGGTACTCAGGTTACCGTGTCTTCCGGTCGT;

[0099] Linker is shown as SEQ ID NO.16: GGTGGTGGTG GTTCTGGTGG TGGTGG CTCT GGCGGTGGTGGTTCC;

[0100] Fc optimized sequence as SEQ ID NO.As shown in Figure 17: GCTTCCACTAAAGGTCCGTCTGTT TTCCCGCTGGCCCCATGTTCTCGTTCCACCTCTGAATCCACTGCAGCACTGGGTTGTCTGGTAAAGGACTACTTTCCGGAGCCTGTTACGGTAAGCTGGAACTCTGGCGCTCTGACCTCTGGTGTGCATACTTTCCCTGCTGTACTGCAGAGCTCTGGCCTGTATTCTCTGTCCTCCGTTGTTACTGTTCCAAGCTCCTCTCTGGGTACTAAAACCTACACCTGTAACGTGGACCACAAACCGTCTAACACCAAAGTTGACAAACGTGTCGAATCCAAATATGGTCCGCCGTGCCCGTCTTGTCCAGCGCCGGAGTTTCTGGGTGGCCCGAGCGTTTTCCTGTTCCCACCAAAGCCGAAAGATACTCTGATGATCTCCCGTACTCCGGAAGTTACCTGCGTTGTTGTGGATGTTAGCCAGGAAGATCCGGAGGTTCAGTTCAACTGGTACGTAGACGGTGTTGAGGTTCACAACGCTAAAACTAAACCACGTGAGGAACAATTCAACTCTACTTACCGTGTTGTTCGTGTACTGACGGTTCTGCATCAGGACTGGCTGAACGGTAAAGAGTACAAGTGCAAAGTGTCCAACAAAGGCCTGCCGTCCAGCATCGAAAAGACTATCAGCAAAGCGAAGGGCCAGCCACGTGAACCGCAGGTTTACACTCTGCCGCCATCCCAGGAGGAAATGACCAAAAACCAGGTGTCTCTGACCTGTCTGGTGAAAGGTTTTTATCCGTCTGACATCGCAGTAGAGTGGGAAAGCAACGGTCAGCCTGAAGACAACTATAAAACCACCCCACCGGTTCTGGACAGCGACGGTAGCTTCTTCCTGTATAGCCGCCTGACTGTGGACAAGAGCCGCTGGCAGGAAGGCAACGTTTTCTCTTGCAGCGTTATGCACGAAGCTCTGCACAACCACTACACCCAAAAATCCCTGTCCCTGTCCCCAGGCAAG;.

[0101] Restriction site: NcoI / XhoI, the synthesis expression vector is pET-28a, the nanobody-Fc-pET-28a plasmid is transferred into the expression strain BL21 (DE3) for expression, IPTG is induced to express, the bacteria are obtained, the bacteria are ultrasonically broken, and the protein is obtained after nickel column affinity purification (balance solution: 20mM Tris and 150mM NaCl, pH8.0; elution solution: 20mM Tris, 150mM NaCl and 100mM imidazole, pH8.0;), the electrophoresis results of the purified antibody Figure 3 .

[0102] Example 2

[0103] 1. Tissue factor nanobody SPR affinity detection

[0104] (1) Conjugation of tissue factor nanobodies

[0105] Nanobody 1 and Nanobody 2 were diluted to 20 μg / ml with sodium acetate (pH 4.5) and amino coupling was performed on a BiacoreX100 instrument using the Manual Run mode. After successful amino coupling of the nanobody, tissue factor protein was detected.

[0106] (2) Tissue factor nanobody SPR affinity detection

[0107] The tissue factor protein (source is the same as the antigen used to immunize alpacas) was diluted with buffer to different concentrations (3.125μg / ml, 6.25μg / ml, 12.5μg / ml, 25μg / ml, 50μg / ml) and injected for 120s, dissociated for 600s, and then affinity tested using the program. The results are as follows Figure 4 and 5 :The affinity constants of tissue factor nanobodies are KD = 2.05 × 10 -9 ; KD = 2.26 × 10 -9 .

[0108] 2. The expression of tissue factor protein on the membrane of pancreatic cancer cells was detected by Western blotting using tissue factor nanobody fused with Fc.

[0109] Pancreatic cancer cells (1×10 6), collect cells by centrifugation, wash with PBS to remove residual serum, lyse with cell lysate, take 20 μL of cell lysate for SDS-PAGE electrophoresis, stop electrophoresis when bromophenol blue runs to the bottom of the gel, transfer to mold, block with 5% skim milk powder for 1 hour, incubate with tissue factor nanobody and Fc fusion protein as primary antibody for 1 hour, incubate for 1 hour, wash the membrane with PBST, incubate with HRP-labeled Fc as secondary antibody at room temperature for 1 hour, wash three times with PBST. Develop, and the results show that the molecular weight of tissue factor protein is 27kDa.

[0110] Example 3

[0111] Detection of tissue factor expression on the surface of pancreatic cancer cells by tissue factor nanoantibody

[0112] Pancreatic cancer cells (2×10 5 ) were inoculated into a culture dish with a coverslip and cultured for 2 days. After observing that the cells on the coverslip are growing well under an inverted microscope, mark the upper left corner of the coverslip with a glass pen to avoid reversing the front and back in subsequent operations. Place the coverslip in formaldehyde fixative and fix at 4 degrees for 10 minutes. Wash three times with PBS, being careful not to pour the PBS solution directly on the cell surface of the coverslip to avoid causing a large number of cell losses. Gently absorb the PBS on the coverslip with filter paper without damaging the cell layer; add 100 μL FITC-labeled tissue factor nanobody ( FITC rapid labeling kit labeled tissue factor nanobody) was dropped on the cover slip and incubated at 37 degrees for 30 minutes; washed three times with PBS and blotted with filter paper; washed three times with PBS and blotted with filter paper, and a drop of liquid paraffin was dropped on the slide. Then the cover slip was placed face down on the slide and observed under a fluorescence microscope. The results are shown in Figure 7 and Figure 8 , tissue factor nanoantibodies can recognize tissue factor on the surface of pancreatic cancer cells.

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nanobody targeting tissue factor, characterized in that: The nanobody comprises: nanobody 1 having an amino acid sequence as shown in SEQ ID NO.1 and / or nanobody 2 having an amino acid sequence as shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the Nanobody according to claim 1, characterized in that The nucleic acid molecule comprises: a nucleic acid molecule 1 whose nucleotide sequence is shown as SEQ ID NO.3 and / or a nucleic acid molecule 2 whose nucleotide sequence is shown as SEQ ID NO.

4.

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

4. The expression vector according to claim 3, characterized in that The basic vector of the expression vector is pET-28a vector.

5. A host bacteria containing the expression vector according to claim 3 or 4.

6. The host bacteria according to claim 5, characterized in that The host bacteria is Escherichia coli BL21 (DE3).

7. Use of the nanobody according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3 or 4, or the host bacteria according to claim 5 or 6 in the preparation of a reagent for detecting tissue factor expression.

8. The use according to claim 7, characterized in that: The samples tested include tumor tissue or tumor cells.

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