Disulfide bond isomerase single-domain antibody as well as preparation method and application thereof

Abstract

The invention discloses a disulfide bond isomerase single-domain antibody as well as a preparation method and application thereof, the disulfide bond isomerase single-domain antibody comprises a variable region, and the variable region comprises CDR1, CDR2 and CDR3; the amino acid sequences of CDR1-CDR3 of the disulfide bond isomerase single-domain antibody are sequentially shown as SEQ ID NO: 15, SEQ ID NO: 16 and SE Q ID NO: 17, and the CDR1-CDR3 are defined according to an IMGT definition scheme. The single-domain antibody provided by the invention can inhibit PDI activity, significantly reduce platelet aggregation and thrombosis, and does not affect the physiological hemostasis function.

Description

Technical Field

[0001] The present invention belongs to the field of antibody technology, and specifically relates to a disulfide isomerase single-domain antibody and a preparation method and application thereof. Background Art

[0002] The protein disulfide isomerase (PDI) family is a class of oxidoreductases widely distributed in the endoplasmic reticulum (ER) of mammalian cells. Their core function is to regulate the correct folding of proteins by catalyzing the formation, cleavage, and rearrangement of disulfide bonds within and between proteins. This family includes members such as PDI, ERp57, ERp5, and ERp72. PDI, the first member of the family to be identified, possesses typical structural features and catalytic activity. While primarily localized in the ER, small numbers of PDIs are also present in the nucleus, cytoplasm, mitochondria, plasma membrane, and extracellular space.

[0003] Single-domain antibodies are composed of a single domain and can be more easily designed to target specific targets, such as coagulation factors or receptors on the platelet surface. This can reduce off-target effects and improve the safety of treatment. As the first approved single-domain antibody drug, caplucizumab specifically targets the A1 domain of von Willebrand factor (VWF) through a bivalent binding mode, effectively blocking the interaction between VWF and the platelet glycoprotein Ib-IX-V complex, thereby inhibiting VWF-mediated platelet adhesion and microthrombosis. This precise targeting strategy significantly reduces the risk of treatment-related bleeding and provides a new paradigm for antithrombotic therapy. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a disulfide isomerase single-domain antibody that can inhibit PDI activity, significantly reducing platelet aggregation and thrombosis without affecting physiological hemostasis.

[0005] The invention also provides a recombinant protein.

[0006] The present invention also provides a biomaterial related to the disulfide isomerase single-domain antibody described in the first aspect of the present invention or the recombinant protein described in the second aspect.

[0007] The present invention also provides a conjugate.

[0008] The present invention also provides a solid phase carrier.

[0009] The present invention also provides an application.

[0010] The present invention also provides a product.

[0011] The present invention also provides a method for preparing the disulfide isomerase single domain antibody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect.

[0012] According to a first aspect of the present invention, a disulfide isomerase single domain antibody is provided, wherein the disulfide isomerase single domain antibody comprises a variable region, wherein the variable region comprises CDR1, CDR2 and CDR3;

[0013] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the CDR1 to CDR3 are defined according to the IMGT definition scheme; or

[0014] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Kabat definition scheme; or

[0015] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Chothia definition scheme; or

[0016] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and the CDR1 to CDR3 are defined according to the Contact definition scheme.

[0017] In some embodiments of the present invention, the amino acid sequence of the disulfide isomerase single domain antibody comprises:

[0018] a1) SEQ ID NO: 14; or

[0019] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 14 and having the same function as the protein shown in SEQ ID NO: 14; or

[0020] a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% identical to SEQ ID NO: 14 and has the same function as the protein shown in SEQ ID NO: 14.

[0021] In some embodiments of the present invention, the disulfide isomerase single domain antibody further comprises a constant region.

[0022] According to a second aspect of the present invention, a recombinant protein is provided, comprising: the disulfide isomerase single domain antibody as described in the first aspect of the present invention; and

[0023] Optional tag sequence to facilitate expression and / or purification.

[0024] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, and a FLAG tag.

[0025] According to the third aspect of the present invention, a biomaterial related to the disulfide isomerase antibody according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention is provided, wherein the biomaterial comprises at least one of h1) to h8):

[0026] h1) a nucleic acid molecule encoding the disulfide isomerase single domain antibody according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention;

[0027] h2) an expression cassette comprising the nucleic acid molecule described in h1);

[0028] h3) a vector comprising the nucleic acid molecule described in h1);

[0029] h4) a vector comprising the expression cassette described in h2);

[0030] h5) a transgenic cell line comprising the nucleic acid molecule described in h1);

[0031] h6) a transgenic cell line comprising the expression cassette described in h2);

[0032] h7) a transgenic cell line comprising the vector described in h3);

[0033] h8) A transgenic cell line comprising the vector described in h4).

[0034] In some embodiments of the invention, the transgenic cell line does not comprise reproductive material.

[0035] In some embodiments of the present invention, the nucleic acid molecule encoding the disulfide isomerase single domain antibody according to the first aspect of the present invention comprises a nucleic acid molecule encoding the variable region of the disulfide isomerase single domain antibody according to the first aspect of the present invention.

[0036] According to a fourth aspect of the present invention, a conjugate is provided, comprising: at least one of the disulfide isomerase single domain antibody according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention;

[0037] and a coupling portion comprising at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein.

[0038] In some embodiments of the present invention, the detectable label is a fluorescent or luminescent label.

[0039] In some preferred embodiments of the present invention, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.

[0040] In some embodiments of the present invention, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188.

[0041] In some embodiments of the present invention, the drug is other drugs for preventing or treating thrombotic diseases (such as antiplatelet aggregation drugs: aspirin, clopidogrel, ticagrelor, tirofiban, cilostazol or dipyridamole, etc.).

[0042] In some embodiments of the present invention, the thrombotic disease comprises arterial thrombotic disease, venous thrombotic disease and microvascular thrombotic disease.

[0043] In some embodiments of the present invention, the arterial thrombotic disease includes coronary heart disease, cerebral infarction, lower limb artery thrombosis, mesenteric artery thrombosis, and renal artery thrombosis.

[0044] In some embodiments of the present invention, the venous thrombotic disease comprises deep vein thrombosis (DVT), pulmonary embolism, portal vein thrombosis, and intracranial venous sinus thrombosis.

[0045] In some embodiments of the present invention, the microvascular thrombotic disease comprises disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

[0046] According to the fifth aspect of the present invention, a solid phase carrier is provided, the surface of which is coupled with the disulfide isomerase single domain antibody described in the first aspect of the present invention and / or the recombinant protein described in the second aspect.

[0047] According to the sixth aspect of the present invention, the use of the disulfide isomerase single-domain antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect, the biomaterial described in the third aspect, the conjugate described in the fourth aspect, and / or the solid phase carrier described in the fifth aspect in the preparation of a product is proposed.

[0048] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0049] In some preferred embodiments of the present invention, the drug has at least one of the functions i1) to i2):

[0050] i1) Inhibit platelet aggregation;

[0051] i2) Prevention and / or treatment of thrombotic diseases.

[0052] In some preferred embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j2):

[0053] j1) detecting the presence or level of disulfide isomerase protein in the sample;

[0054] j2) Diagnosis of thrombotic diseases.

[0055] In some embodiments of the present invention, the thrombotic disease comprises arterial thrombotic disease, venous thrombotic disease and microvascular thrombotic disease.

[0056] In some embodiments of the present invention, the arterial thrombotic disease includes coronary heart disease, cerebral infarction, lower limb artery thrombosis, mesenteric artery thrombosis, and renal artery thrombosis.

[0057] In some embodiments of the present invention, the venous thrombotic disease comprises deep vein thrombosis (DVT), pulmonary embolism, portal vein thrombosis, and intracranial venous sinus thrombosis.

[0058] In some embodiments of the present invention, the microvascular thrombotic disease comprises disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

[0059] According to a seventh aspect of the present invention, a product is provided, comprising at least one of k1) to k4):

[0060] k1) the disulfide isomerase single domain antibody according to the first aspect of the present invention;

[0061] k2) the recombinant protein according to the second aspect of the present invention;

[0062] k3) the conjugate according to the fourth aspect of the present invention;

[0063] k4) The solid phase carrier described in the fifth aspect of the present invention.

[0064] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0065] In some preferred embodiments of the present invention, the drug has at least one of the functions i1) to i2):

[0066] i1) Inhibit platelet aggregation;

[0067] i2) Prevention and / or treatment of thrombotic diseases.

[0068] In some preferred embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j2):

[0069] j1) detecting the presence or level of disulfide isomerase protein in the sample;

[0070] j2) Diagnosis of thrombotic diseases.

[0071] According to the eighth aspect of the present invention, a method for preparing the disulfide isomerase single domain antibody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention is proposed, which is obtained by culturing the transgenic cell line described in the third aspect of the present invention.

[0072] The present invention has at least the following beneficial effects:

[0073] The present invention screened an anti-PDI single-domain antibody using phage display technology and verified its in vitro anti-platelet aggregation and in vivo inhibitory activity against FeCl3-induced thrombosis. The PDI single-domain antibody provided by the present invention can inhibit the reductase activity of PDI in vitro with greater potency than the positive control RL90, significantly inhibiting platelet aggregation induced by AA, ADP, and PAF. In vivo, it significantly reduced the weight of FeCl3-induced carotid artery thrombi in rats without prolonging tail bleeding time, suggesting that it selectively intervenes in pathological thrombosis without interfering with basic hemostasis. This invention provides key experimental evidence for the therapeutic strategy of anti-PDI single-domain antibodies in thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0075] Figure 1 This is a flowchart of the experiment for screening single domain antibodies using phage display technology in Example 1 of the present invention;

[0076] Figure 2 Figure 1 is a diagram showing the identification results of the purified PDI protein in Example 1 of the present invention; wherein A is SDS-PAGE analysis and B is Western blot detection;

[0077] Figure 3 Graph showing the specific phage enrichment results in Example 1 of the present invention; wherein A represents the phage titer determination, and B represents the enrichment results after three rounds of screening;

[0078] Figure 4 This is a diagram showing the screening results of positive monoclonal clones screened by phage ELISA in Example 1 of the present invention;

[0079] Figure 5 The figure is a graph showing the agarose gel electrophoresis results of the positive monoclonal phage in Example 1 of the present invention; wherein, lane M is a marker, and lanes 1 to 9 are bands of PCR products of the positive monoclonal phage colony;

[0080] Figure 6 This is an amino acid sequence alignment analysis diagram in Example 1 of the present invention;

[0081] Figure 7 This is a comparison diagram of the CDRs of the sequences analyzed by the IMGT website in Example 1 of the present invention;

[0082] Figure 8 This is a diagram showing the electrophoresis identification results of the PCR products of the target gene in Example 1 of the present invention;

[0083] Figure 9 This is the plasmid spectrum of SdAb in Example 1 of the present invention;

[0084] Figure 10 This is a diagram showing the electrophoresis identification results of the positive colony PCR products in Example 1 of the present invention;

[0085] Figure 11 Graph showing the expression verification results of the PDI single-domain antibody in Example 1 of the present invention; wherein, M is a marker, lane 1 represents bacteria collected under 16°C induction conditions, lane 3 represents the supernatant after ultrasonic disruption of bacteria at 16°C and centrifugation, lane 4 represents the precipitate after ultrasonic disruption of bacteria at 16°C and centrifugation, lane 5 represents the supernatant after ultrasonic disruption of bacteria at 30°C and centrifugation, and lane 6 represents the precipitate after ultrasonic disruption of bacteria at 30°C and centrifugation;

[0086] Figure 12 Figure for the purification and identification results of the PDI single-domain antibody in Example 1 of the present application; wherein, A is the verification result of the elution of the target protein, lane M is a marker, lane 1 is supernatant, lane 2 is flow-through, lanes 3-7 are 50-100 mM imidazole elution of impure proteins, and lanes 8-10 are 500 mM imidazole elution of target proteins; B is a result graph for identifying the single-domain antibody by Western blot;

[0087] Figure 13 Figure for the antigen binding capacity verification results of the PDI single-domain antibody in Example 1 of the present application; wherein, A is a result graph of SDS-PAGE, lane M is a marker, lane 1 is a 10 μg PDI protein loading amount, and lane 2 is a 20 μg PDI protein loading amount; B is a Western blot result, the band after ECL color development of PDI protein incubated with PDI single-domain antibody and HRP-protein A; C is an ELISA detection of binding capacity, BSA, coated with 5% BSA; PDI, coated with PDI protein;

[0088] Figure 14 Figure for the results of using the Octet system to determine the affinity of the PDI single-domain antibody to PDI protein in Example 2 of the present application;

[0089] Figure 15 Figure for the detection results of platelet LDH release after treatment with the PDI single-domain antibody in Example 3 of the present application; wherein, Con is a blank control group, Veh is a solvent control group, SdAb is a PDI single-domain antibody, and Max is a maximum enzyme activity control, which is standardized to 100%; compared with Max, *P≤0.05;

[0090] Figure 16 Inhibition curve of PDI single-domain antibody of different concentrations on PDI activity in Example 3 of the present application;

[0091] Figure 17 Figure for the detection results of the inhibition ability of the PDI single-domain antibody on PDI enzyme activity in Example 3 of the present application; wherein, A is the inhibition rate, and B is the half maximal inhibitory concentration IC 50 ;

[0092] Figure 18 Figure for the in vitro verification results of the anti-platelet aggregation of the PDI single-domain antibody in Example 3 of the present application; wherein, A, C, and E are the inhibition rates of PDI single-domain antibodies of different concentrations on ADP, AA, and PAF-induced platelet aggregation, respectively; B, D, and F are the half maximal inhibitory concentrations IC 50 ;

[0093] Figure 19This is a graph showing the test results of tail bleeding time in rats after treatment with the PDI single domain antibody in Example 3 of the present invention;

[0094] Figure 20 3. This is a graph showing the detection results of carotid artery thrombosis in rats after treatment with the PDI single domain antibody in Example 3 of the present invention; wherein, A shows the morphology of the rat carotid artery after dissection, and B shows the effect of the PDI single domain antibody on the weight of FeCl3-induced carotid artery thrombosis in rats. DETAILED DESCRIPTION

[0095] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0096] Example 1 Screening, expression and purification of disulfide isomerase single domain antibodies

[0097] In this example, a commercial phage display library was used to screen positive colonies with high affinity for PDI protein and sequenced to obtain positive monoclonal phage sequences. Subsequently, the PDI single domain antibody plasmid was constructed, expressed, purified, and identified. The experimental flow chart of phage display technology for screening single domain antibodies is shown in the figure. Figure 1 The specific methods and results are as follows:

[0098] 1. Screening of single domain antibodies

[0099] 1) Expression, purification and identification of PDI protein:

[0100] 1 μL of the preserved PDI recombinant plasmid (the plasmid backbone is pTriEx-4 Neo Vector, and the target gene fragment is the mRNA fragment of the 65-1591 region in the NCBI database NM_000918.4 (P4HB)) was transformed into BL21 (DE3) competent cells, and 200 μL of the bacterial solution was spread on LB solid medium containing antibiotics and incubated in a 37°C constant temperature incubator for 16 hours. The next day, a monoclonal colony with regular morphology was picked and inoculated into 5 mL of LB liquid medium, and after shaking culture at 37°C for 5 hours, it was transferred to 200 mL of fresh culture medium. When the OD 600When the value reaches 0.6, add 1 mL of IPTG to a final concentration of 0.8 mM, and continue induction at 16°C with shaking for 6 hours. Collect the cells by centrifugation at 8,000 × g for 10 minutes at 4°C, wash with PBS, and repeat the centrifugation. Resuspend the cells in 20 mL of PBS and disrupt them by sonication on ice at 60% power, using a #6 horn, for 3 seconds on, 6 seconds off, for a total of 15 minutes. After disruption, centrifuge at 12,000 × g for 10 minutes, and collect the supernatant to obtain the crude PDI protein solution.

[0101] The His-tagged PDI fusion protein was purified by nickel column affinity chromatography, and the protein concentration of the PDI fusion protein was determined using a BCA protein quantification kit. The purified PDI fusion protein was then identified by SDS-PAGE and Western blot based on the protein concentration. The results are shown in Figure 2. Figure 2 shown.

[0102] Depend on Figure 2 It can be seen that the purified product presents a single target band in the range of 55-70kDa ( Figure 2 A), the target protein has a specific signal at the expected molecular weight position (theoretical value 57kDa) ( Figure 2 B) PDI protein was mainly present in the supernatant, indicating that it was efficiently expressed in a soluble form and its molecular weight was consistent with the theoretical value.

[0103] 2) Screening of antibody library using microplate screening method:

[0104] The helper phage M13 was co-incubated with a frozen antibody library (purchased from Source Bioscience, human domain antibody library (Dab library)) to obtain a phage display antibody library. The phage display antibody library was then incubated with PDI protein in a microplate for three rounds of "adsorption-elution-amplification". TG1 was used as the receptor bacterium. The specific method is as follows:

[0105] ① Dilute PDI protein to 10 μg / mL in carbonate coating solution. Add 100 μL per well to the ELISA plate and incubate overnight at 4°C (the antigen concentration for the next two rounds is 5 μg / mL). The next day, discard the coating solution and wash the plate three times with 250 μL of PBS per well. Pat the plate dry each time to remove any remaining liquid. Then, add 200 μL of 5% BSA and block at 37°C for 2 hours. Discard the blocking solution and wash each well five times with 200 μL of PBST and once with 200 μL of PBS. Add 100 μL of the freshly prepared antibody library to each well and incubate at 37°C for 2 hours. Discard any phage in the wells and add 200 μL of PBST per well. Wash the plate five times to remove unbound phage (wash 10 times in the next two rounds). 100 μL of trypsin elution solution was added to each well and the well was allowed to stand at room temperature for 15 min, during which time the well was pipetted twice to elute the specifically bound phages to obtain the first round of specific phages.

[0106] ② Streak the recipient bacteria TG1 on an M9 plate and incubate at 37°C overnight. Pick a single TG1 colony and place it in 5 mL of 2×TY. After diluting it 100-fold, grow it at 37°C, 250 rpm overnight until the OD 600 =0.5. Add 30mL of the above culture medium to the eluted phage and incubate in a 37°C water bath for 1h. Then centrifuge at 4°C, 3200g for 5min. Resuspend the precipitate with 1mL of 2×TY, spread it onto a TYE culture dish supplemented with 4% (wt / vol) glucose and 100μg / mL ampicillin, and incubate it overnight at 37°C. At the same time, use the gradient dilution method on the TYE plate to check the titer of the eluted phage. The next day, scrape the plaque into a test tube containing 5mL of 2×TY culture medium and dilute it with 500mL of 2×TY medium supplemented with 4% (wt / vol) glucose and 100μg / mL ampicillin. Grow in a 2L conical flask at 37°C, 250rpm to OD 600 = 0.5. Phages were purified and precipitated with PEG and the resulting phages were used for two subsequent rounds of repeated screening.

[0107] Among them, during the three rounds of "adsorption-elution-amplification" mentioned above, the titers of input phage and output phage in each round were tested, and the enrichment factor was calculated accordingly (the ratio of output phage / input phage titer in the next round divided by the ratio in the first round). The results are as follows Figure 3 and as shown in Table 1.

[0108] Table 1 Enrichment of specific phages

[0109]

[0110] Combine Figure 3 As shown in Table 1, the input phage titer remained constant during the three rounds of screening (5×10 12pfu / mL), while the output phage titer was significantly improved round by round: 2.5 x 10 6 pfu / mL (output / input titer ratio = 5.0 x 10 -7 ) in the first round, 1.2 x 10 8 pfu / mL (output / input titer ratio = 2.4 x 10 -5 , 48-fold enrichment of specific phage), and 1.8 x 10 8 pfu / mL (output / input titer ratio = 3.6 x 10 -5 , 72-fold enrichment of specific phage) in the third round; compared with the first round, the output / input titer ratio was improved by 72-fold in the third round, indicating that the specific phage clone was effectively enriched in the screening process Figure 3 B), and the enrichment fold was only 1.5 times more than that in the second round after increasing the number of washing times in the third round, so the fourth round of binding and elution was not performed.

[0111] After the above-mentioned 3 rounds of screening, positive monoclonal phage was screened by ELISA to test the PDI protein binding capacity of individual colonies. 80 monoclonal colonies were picked and inoculated into 2 x TY medium containing ampicillin and glucose, supplemented with 100 μg / mL ampicillin and 4% (wt / vol) glucose, and cultured at 37°C for 3 hours. After centrifugation for 1 hour after infection with helper phage, the precipitate was resuspended in medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1% (w / v) glucose, and grown at 25°C, 250 rpm for 16-24 hours. The supernatant was collected by centrifugation, and the specific phage was collected for standby. The ELISA plate was coated with PDI protein and blocked overnight, and the 80 groups of specific phage collected above were mixed with MT buffer (PBS buffer supplemented with 5% BSA and 2% Tween 20) and added to the ELISA plate, followed by HRP-anti M13 antibody, color development, and reading at 450-650 nm on the enzyme label instrument. The negative group was directly blocked overnight without coating PDI protein, and the results are shown in Figure 4 .

[0112] The ELISA results of Figure 4 were analyzed, and the OD ratio of the positive group (PDI group) to the negative group (BSA group) was greater than 2.5 to determine the positive monoclonal phage. The top 20 colonies with the highest ratio were selected from the positive monoclonal phage for expansion culture, and 9 colonies were randomly selected for PCR identification. The PCR product bands of the 9 colonies are shown in Figure 5The above 20 colonies were sent to Shanghai Biotech Co., Ltd. for sequencing (primer 5'-CAGGAAAC AGCTATGACCATG-3' (SEQ ID NO: 1)). The sequencing results were analyzed by DNAMAN. The results are shown in the figure. Figure 6 and Figure 7 shown.

[0113] Depend on Figure 6 It was found that among the 20 clones, the G5 sequence was repeated 10 times (50%), the 44 sequence was repeated 6 times (30%), and the 41, 42, A1, and E2 sequences were repeated once each (5% each).

[0114] Depend on Figure 7 It can be seen that after dividing the CDR region using the IMGT database (https: / / www.imgt.org / ), it was found that the CDR3 of G5 has a 12-amino acid sequence, which is longer than other sequences. The six sequence colonies were selected, expanded, and retested by ELISA. The results showed that the OD of the G5 sequence was 450 Based on the cloning frequency (G5 had the highest proportion) and ELISA retest results, the G5 sequence was selected for subsequent experiments.

[0115] 2. Expression and Purification of Single Domain Antibodies

[0116] 1) Construction of single-domain antibody expression vector:

[0117] ①Synthesis of single domain antibody genes:

[0118] To clone the target gene sequence with a 6×His tag at the C-terminus into the pET-28(+) vector, NcoI (5' end) and XhoI (3' end) restriction sites were first introduced at both ends of the target sequence. Twelve primers (Table 2) were designed to amplify the target gene in segments: primers 1-2 covered the 5' NcoI site and the initiation region, primers 3-10 synthesized the main gene segment, and primers 11-12 introduced the XhoI site, the stop codon, and the 3' homology arm. 20-40 bp of homologous overlapping sequences were designed at the end of each primer pair to achieve seamless splicing between fragments. The matching of the overlapping regions of each fragment and the correct reading frame were then verified by simulation using Snap Gene software.

[0119] Table 2 Primers used to synthesize target genes

[0120]

[0121]

[0122] The primers were synthesized by Beijing Genki Biological Technology Co., Ltd. After the synthesis of the primers, the target gene was synthesized by a PCR reaction. A 50 μL system was used, the reagents to be prepared were thawed on ice, and the components in Table 3 were added in proportion on a clean bench. After the reagents were added, the PCR instrument was started, the program was created, and the denaturation, annealing, and extension parameters were set according to Table 4, and the running was started.

[0123] Table 3 PCR reaction system

[0124]

[0125] Table 4 PCR reaction conditions

[0126]

[0127] ②Amplification of single-domain antibody gene:

[0128] The target gene was amplified by PCR. A 50 μL system was used, the reagents to be prepared were thawed on ice, and the following components in Table 5 were added in proportion on a clean bench. After the reagents were added, the PCR instrument was started, the program was created, and the reaction conditions were referred to Table 4.

[0129] Table 5 PCR reaction system

[0130]

[0131] ③Recovery of single-domain antibody gene: After the PCR reaction was completed, the PCR product was separated by electrophoresis using a 2% agarose gel, and the target band size was about 500 bp (as shown in Figure 8 After electrophoresis, the gel corresponding to the molecular weight size was cut off, and the Biyun Tian DNA gel recovery kit was used for gel recovery; then the target gene and the pET-28a(+) vector were treated with restriction endonuclease NcoI and XhoI and gel recovery was performed, obtaining the single-domain antibody target gene and the vector with complementary cohesive ends.

[0132] ④Connection of single-domain antibody gene and vector:

[0133] The single-domain antibody target gene recovered in step ③ was connected with the pET-28a(+) vector, and the connection system is shown in Table 6. The reagents in the table were added in a sterile centrifuge tube, and then 50℃ water bath for 25 min, transformation. After 2-3 min, the temperature was reduced, and the bacterial solution was spread on the LB plate containing kanamycin, and incubated at 37℃ overnight. The recombinant plasmid SdAb (plasmid map as shown in Figure 9 ) was constructed.

[0134] Table 6 Recombination reaction system

[0135]

[0136] ⑤ Identification of single domain antibody recombinant plasmid:

[0137] Pick a single colony from the overnight plate in step ④ and perform colony PCR using primers 1 and 12 in Table 5. Identify positive clones by agarose gel electrophoresis. The results are as follows: Figure 10 As shown; 5 positive colonies were randomly selected and placed in 1.5 mL centrifuge tubes. They were cultured overnight at 37°C in a shaker. Plasmids were extracted from the overnight bacterial culture using the kit protocol. The obtained single-domain antibody plasmid was sequenced to ensure sequence accuracy.

[0138] Depend on Figure 10 It can be seen that all five monoclonal clones showed a single clear band at approximately 500 bp.

[0139] These results demonstrate the successful construction of the PDI single-domain antibody recombinant plasmid SdAb, which can be used for subsequent protein expression. The amino acid sequence of the constructed PDI single-domain antibody is as follows: MAQVQLLESGGGLVQPGGSLRLSCAASGFKISH KSMGWVRQAPGKGLEWVSAIRARGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCATVEVREHQKYLKFWGQGTLVTVSSAAAHHHHHH* (SEQ ID NO: 14). The single-domain antibody has a total of 132 amino acids and a size of 14.5 kDa.

[0140] The CDR1, CDR2, and CDR3 of the three variable regions of the PDI single domain antibody defined by different definition schemes are shown in Table 7.

[0141] Table 7 CDR1, CDR2, and CDR3 defined by different definition schemes

[0142]

[0143] 2) Isopropylthiogalactoside (IPTG)-induced expression and identification of recombinant proteins:

[0144] The recombinant plasmid SdAb obtained in step 1) was transformed into Escherichia coli BL21 (DE3), and IPTG was used to induce the expression of the recombinant protein. Two sets of induction conditions were set at 16°C and 30°C, respectively. The specific experimental method is as follows:

[0145] Pick a single colony from the transformation plate and inoculate it into a test tube containing 5 mL of LB medium (containing 50 μg / mL kanamycin) and culture it at 37°C and 220 rpm overnight. The next day, transfer it to 100 mL of LB medium (containing 50 μg / mL kanamycin) at a ratio of 1:100 and culture it at 37°C and 220 rpm until the bacterial OD reaches 0. 600The pH value is 0.5-0.8. For the negative control sample (without IPTG): Place 1 mL of the culture in a 1.5 mL centrifuge tube and centrifuge at 10,000 × g for 2 minutes at room temperature. Discard the supernatant and resuspend the pellet in 100 μL TBS. Add 25 μL of 5× SDS-PAGE protein loading buffer, incubate at 100°C in a metal bath for 10 minutes, and freeze at -20°C. Add IPTG to the remaining culture to a final concentration of 0.2 mM, then adjust the temperature to 16°C or 30°C and continue shaking for 18 hours to induce expression of the fusion protein. For the positive control sample (with IPTG): Place 1 mL of the induced culture in a 1.5 mL centrifuge tube and centrifuge at 10,000 × g for 2 minutes at room temperature. Discard the supernatant and resuspend the pellet in 100 μL TBS. Add 25 μL of 5× SDS-PAGE protein loading buffer, incubate at 100°C in a metal bath for 10 minutes, and freeze at -20°C. The remaining culture was centrifuged at 4°C, 4000×g for 10 min, and the supernatant was discarded. The cells were resuspended in TBS, and PMS F was added to the resuspension at a final concentration of 1 mM. Ultrasonic disruption was performed, and the precipitate and supernatant were collected after centrifugation. Analyzed by 12% SDS-PAGE and stained with Coomassie Brilliant Blue for band development. The results were as follows: Figure 11 shown.

[0146] Depend on Figure 11 As can be seen, the target band appeared at 14.5kDa in both induction conditions. In the 30°C induction group, the target protein was mainly present in the precipitate (lane 6), with only a small amount detected in the supernatant (lane 5). In the 16°C induction group, the opposite was true, with a significantly higher proportion of the target protein in the supernatant (lane 3) and less precipitate (lane 4). The soluble protein yield in the 16°C induction group was higher than that in the 30°C group. Because the inclusion body protein (precipitate) requires complex refolding and has a low activity recovery rate, the 16°C low-temperature induction condition was ultimately determined to be the optimal condition for achieving efficient expression of soluble single-domain antibodies.

[0147] 3) Purification of single domain antibodies:

[0148] Purification was performed using a low pressure chromatography system, as follows:

[0149] ① Connect the Ni-NTA affinity chromatography column (pre-filled with 5 mL of medium) to the low-pressure chromatography system and pre-equilibrate it with binding buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) at a flow rate of 1 mL / min. 280 The value reaches the baseline.

[0150] ② Perform preliminary elution with elution buffer containing 50 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of the effluent three times in sequence.

[0151] ③ Perform preliminary elution with elution buffer containing 100 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of effluent twice.

[0152] ④ Perform preliminary elution with elution buffer containing 500 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of effluent three times in sequence.

[0153] ⑤ Transfer 40 μL of the flow-through and each effluent into a 1.5 mL centrifuge tube and add 10 μL of 5× SDS-PAGE protein loading buffer to each tube. Incubate in a metal bath at 100°C for 10 minutes, then freeze at -20°C. Store the remaining effluent at 4°C.

[0154] ⑥ The components collected by the above gradient elution were analyzed by SDS-PAGE. The results were as follows: Figure 12 As shown in A.

[0155] Depend on Figure 12 As shown in A, lane 2 (flow-through): no target protein band was seen, indicating that the His-tagged protein was effectively bound to the column matrix; lanes 3-5 (50mM imidazole elution): multiple miscellaneous protein bands appeared, but no target band (14.5kDa); lanes 6-7 (100mM imidazole elution): the amount of miscellaneous proteins decreased, but there was still no target band; lanes 8-10 (500mM imidazole elution): trace miscellaneous bands were visible in lanes 8-9, and lane 10 (500mM imidazole main peak) showed a single clear target band, with a purity of >90%.

[0156] 4) Ultrafiltration concentration of single domain antibodies:

[0157] The eluate collected in step 3) was placed in a dialysis bag and dialyzed against TBS (20 mM Tris, 0.5 M NaCl, pH 8.0) at a ratio of 1:30. The TBS was changed twice every 2 hours. The eluate was then concentrated using a 5 kDa ultrafiltration tube at 3500 × g for 15 minutes until no more cut-off volume was present. The protein concentration after ultrafiltration was determined using a BCA protein quantification kit.

[0158] After the concentrated protein sample was subjected to SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer apparatus (20V, 30min), and then blocked with 5% BSA at room temperature for 2h. Then, diluted primary antibodies (His antibody) were added, reacted at 4℃ overnight, and then washed three times on a shaker with TBST solution. After completion, secondary antibodies were added and incubated at room temperature for 2h. The membrane was exposed to ECL chemiluminescence reagent and developed. The results are shown in the figure. Figure 12 As shown in B.

[0159] Depend on Figure 12As shown in B, a single specific band at 14.5 kDa was detected, confirming that the purified protein was a His-tagged PDI single domain antibody.

[0160] Example 2: Antigen Binding Ability Test of Disulfide Isomerase Single Domain Antibody

[0161] This example verifies the binding ability of the PDI single domain antibody prepared in Example 1 to the PDI protein in vitro. The specific experimental methods and results are as follows:

[0162] 1.Western blot:

[0163] The PDI recombinant protein prepared in Example 1 (10 μg and 20 μg / lane, respectively) was separated by 12% SDS-PAGE. Figure 13 Lanes 1 and 2 in A) were transferred to a PVDF membrane. After blocking for 2 hours, the protein blot was incubated overnight with the single domain antibody prepared in Example 1 (primary antibody, 1:1000 dilution), and then incubated with HRP-protein A (secondary antibody, 1:5000 dilution). ECL color development solution was added to the strips and photographed using an ECL imaging system. The results are shown in Figure 2. Figure 13 As shown in B.

[0164] Depend on Figure 13 B shows that a single specific band appears at 48-63 kDa, confirming that the single domain antibody screened in Example 1 of the present invention can bind to PDI (e.g. Figure 13 B).

[0165] 2. Indirect ELISA:

[0166] The recombinant PDI protein prepared in Example 1 was coated onto a 96-well plate, the PDI single domain antibody screened in Example 1 was used as the primary antibody, and HRP-protein A was used as the secondary antibody. After incubation and color development, the OD value was measured at 450 nm using a microplate reader. The specific steps are as follows:

[0167] (1) Antigen coating: PDI protein solution (5 μg / mL, dissolved in carbonate-bicarbonate coating buffer) was coated into a 96-well plate at a volume of 100 μL per well. A 5% BSA solution was set as a negative control well. After sealing the plate, it was incubated at 4°C overnight (16-18 h). Four replicate wells were set for each sample.

[0168] (2) Blocking: The next day, the coating solution was discarded and 250 μL of blocking buffer (PBST containing 5% BSA, pH 7.4) was added to each well and incubated at 37°C for 2 h.

[0169] (3) Primary antibody binding: PBST wash 4 times (300 μL / well each time, soak for 2 min), pat dry residual liquid. Add 100 μL / well of single-domain antibody solution (5 μg / mL, PBST dilution), 150xg, 37°C horizontal shaking incubation for 45 min.

[0170] (4) Secondary antibody binding: recover the primary antibody solution, repeat the PBST washing step. Add 50 μL HRP-Protein A (1:5000 dilution in PBST) per well, incubate at 37°C for 45 min in the dark.

[0171] (5) Color development reaction: recover the secondary antibody solution, after PBST washing, add 50 μL TMB color development substrate per well, color development at 37°C for 10 min in the dark. Add 50 μL / well 2M sulfuric acid stop solution to stop the reaction, use an enzyme label instrument to measure the 450 nm absorbance, and the results are shown in Figure 13 C.

[0172] As can be seen from Figure 13 C, the OD 450 of the PDI protein coated wells is significantly higher than that of the BSA control wells, and the ratio is greater than 2.5, indicating that the PDI single-domain antibody screened by the application has affinity with PDI protein.

[0173] 3. Determine the real-time interaction kinetic parameters:

[0174] Using a Sartorius Octet molecular interaction instrument (model: R8), the real-time interaction kinetic parameters of the two were determined by bio-layer interferometry (Bio-Layer Interferometry, BLI). This technology is based on the light interference effect of the fiber sensor probe tip: when the PDI protein is fixed on the sensor surface and combined with the PDI single-domain antibody, the change in the thickness of the molecular layer will cause the displacement of the interference light wave form (real-time monitoring with nanometer-level precision), thereby directly obtaining the association rate constant K on , unit: M-1·s -1 (per mole per second) and the dissociation rate constant K off , unit: s -1 (per second), and the equilibrium dissociation constant KD is calculated by the formula KD=K off / K on , unit: M (molar concentration). Among them, K on characterizes the molecular binding efficiency, K off reflects the dissociation rate of the complex, and the KD value comprehensively characterizes the overall affinity of the interaction between the two. By accurately determining the KD value, the interaction strength of the single-domain antibody and the PDI protein can be quantitatively evaluated.

[0175] Using an anti-streptavidin biosensor, PDI protein was immobilized to a response value of 1 nm (mixed with Thermo21312 biotin at a molar ratio of 1:3 and reacted at room temperature for 30 min). Binding and dissociation assays were performed using a gradient dilution of the single domain antibody (500 nM-18.5 nM). The experimental data were fitted and analyzed using a Langmuir model based on a 1:1 stoichiometric relationship (software: Analysis Studio), calculated KD = 4.5 × 10 -7 M, K on =3.1×10 5 M -1 ·s -1 , K off =1.4×10 -1 s -1 , the results are as follows Figure 14 As shown, Figure 14 The results confirmed that the PDI single domain antibody screened by the present invention has a strong affinity with the PDI protein.

[0176] In summary, the present invention successfully screened out single-domain antibodies against PDI for the first time using phage display technology. Through three rounds of "adsorption-elution-amplification" selection, six anti-PDI single-domain antibody sequences were screened from the single-domain antibody library. Among them, the G5 sequence was selected as the research object because of its high sequence repetition rate and ELISA retest results that were better than the other five sequences. A 14.5kDa single-domain antibody was successfully expressed and purified using a prokaryotic expression system (Escherichia coli BL21). Western blot, ELISA, and molecular interaction experiments confirmed its ability to bind to PDI. The PDI single-domain antibody provided by the present invention provides a key guarantee for subsequent experiments and lays the foundation for the development of treatment methods based on anti-PDI single-domain antibodies.

[0177] Example 3 Antithrombotic Activity Test of PDI Single Domain Antibody

[0178] This example uses in vitro and in vivo experiments to test the anti-thrombotic ability of the PDI single domain antibody screened and prepared in Example 1. The specific test methods and results are as follows:

[0179] 1. Determination of platelet lactate dehydrogenase (LDH) release

[0180] The experiment was divided into 6 groups: a blank control group (Con) containing Tyrode's solution, a solvent control group (Veh) containing TBS, an experimental group (SdAb) containing 0.5, 1, and 2 μM single-domain antibodies, and a maximum enzyme activity control group (Max) containing lysis buffer. In a 96-well plate, 230 μL of washed pretreated platelets and 20 μL of the corresponding test sample were added to each group in sequence, mixed evenly, and incubated at 37°C for 5 minutes; LDH release reagent was added and incubated at 37°C for 1 hour, and centrifuged at 400 × g for 5 minutes; 120 μL of the supernatant from each group was transferred to a new 96-well plate, 60 μL of detection solution was added, and the plate was incubated at room temperature in the dark for 30 minutes. The absorbance was measured at a wavelength of 490 nm using a microplate reader; 3 replicates were set up for each group and each experiment, and three independent repetitions were performed. The results are shown in the figure below. Figure 15 shown.

[0181] Platelet lactate dehydrogenase (LDH) is a marker of cell damage, and its release can be used to evaluate the potential toxicity of single-domain antibodies to platelets. By adding lysis buffer to completely destroy the platelet membrane structure, all LDH in the platelets is released, and the maximum enzyme activity benchmark of the experimental system is calibrated. Figure 16 It can be seen that compared with the Con group, the LDH release amounts of the single-domain antibody groups and the Veh group at different concentrations did not show significant differences (P>0.05); compared with the Max group, the LDH release amounts of the single-domain antibody group showed significant differences (P≤0.05), indicating that within the experimental concentration range, the PDI single-domain antibody and its solvent TBS provided by the present invention did not cause platelet membrane structure damage.

[0182] 2. In vitro enzyme inhibitory activity assay

[0183] The ability of single-domain antibodies to inhibit PDI activity was evaluated using the insulin turbidimetric assay. The experiment was divided into eight groups: a background group (only insulin + DTT), a model group (containing PDI protein but no single-domain antibody), an experimental group (containing PDI single-domain antibodies at final concentrations of 0.5, 1, 2, 4, and 8 μM), and a positive group (containing RL90). The experimental system consisted of 80 μL, with a final concentration of 0.04 mg / mL PDI protein and 1 mg / mL insulin. In a 96-well plate, 20 μL of buffer (100 mM potassium dihydrogen phosphate, 1 mM EDTA, pH 7.4) was added to the background group, 10 μL of buffer and 10 μL of PDI protein were added to the model group, 10 μL of each concentration of PDI single-domain antibody and 10 μL of PDI protein were added to the experimental group, and 10 μL of RL90 and 10 μL of PDI protein were added to the positive group. After incubation at 37°C for 30 minutes, 50 μL of insulin and 10 μL of 1.5 mM DTT were added in sequence. The plate was placed in a microplate reader and run at 25°C for 140 minutes. The assay was performed every 10 minutes, with 3 replicates set up each time. Three independent experiments were repeated. The formula for calculating the inhibition of PDI enzyme activity is: Enzyme activity inhibition rate (%) = [1-(OD [单域抗体+PDI+DTT] -OD [DTT] ) / (OD [PDI+DTT] -OD [DTT] )]×100%, the result is as follows Figure 16 and Figure 17 shown.

[0184] The principle of the insulin turbidimetric method is that in the presence of DTT, PDI catalyzes the cleavage of disulfide bonds in insulin molecules through reductase activity, triggering β-chain aggregation and increasing solution turbidity. By detecting the change in transmittance at a wavelength of 630nm, the level of PDI activity can be indirectly reflected. Figure 16 It can be seen that the PDI single domain antibody screened and expressed in the present invention has the ability to inhibit PDI activity, and the ability to inhibit PDI activity increases with increasing concentration. At the same time, the reported PDI monoclonal antibody RL90 was selected as a positive control. Figure 17 The results showed that the inhibitory effect of the single-domain antibody was better than that of RL90; the experiment was repeated three times, and the inhibition rate of the single-domain antibody on PDI activity was calculated by the formula ( Figure 17 A), and draw the half-maximal inhibitory concentration curve ( Figure 17 B), its IC 50 =4.52±0.17μM.

[0185] 3. In vitro Antiplatelet Aggregation Rate Determination

[0186] Rabbit blood was collected and centrifuged to obtain PRP. The remaining plasma in the tube was mixed and centrifuged at 1500g for 10 minutes. The supernatant was taken as platelet-poor plasma (PPP). 215 μL of PRP was mixed with 10 μL of PDI single-domain antibodies of different concentrations (0.25, 0.5, 1, 1.5, 2 μM) and incubated at 37°C for 5 minutes. During the test, 250 μL of PPP was first injected into the cuvette, and the instrument calibration was started. When the screen displayed the OD value of PPP, the PRP that had been incubated for 5 minutes was inserted into the detection channel and the test was started. When a curve appears on the screen, 25 μL of different inducers are added: arachidonic acid (AA) with a final concentration of 1.5 mM; 15 μM adenosine diphosphate (ADP) and 10 nM platelet activating factor (PAF) to induce platelet aggregation in vitro. At the same time, a control group (Con) and an irrelevant protein 5% BS A group are set up to exclude platelet aggregation caused by protein structure. The entire experimental reaction process is carried out on a platelet aggregation instrument. The changes in platelet aggregation during the process are recorded by the instrument for 5 minutes. After the experiment, the instrument automatically calculates the maximum platelet aggregation rate (%), and the results are as follows. Figure 18 shown.

[0187] Depend on Figure 18 It can be seen that the PDI single domain antibody provided by the present invention can significantly inhibit platelet aggregation induced by ADP, AA, and PAF ( Figure 18 A / C / E), half inhibitory concentration IC 50 They were 1.29±0.06μM, 1.09±0.05μM, 1.52±0.02μM ( Figure 19 B / D / F). In the platelet aggregation model induced by AA and ADP, the single domain antibody with a final concentration of 0.5, 1, 1.5, and 2 μg / mL showed significant differences compared with Con (P≤0.05) ( Figure 18 A / B). In the PAF model, the single domain antibody with a final concentration of 1, 1.5, and 2 μM showed significant differences compared with Con (P≤0.05) ( Figure 18 C). In the ADP model, the monoclonal antibody RL90 was used and the results showed that its effect was comparable to that of RL 90 ( Figure 18 A).

[0188] 4. Rat Thrombosis Model to Explore the Effect of PDI Single Domain Antibody on Thrombosis in Vivo

[0189] 1) Animal grouping and tail-cut bleeding time determination:

[0190] The effect of PDI single domain antibody on coagulation function was evaluated by tail bleeding time in rats.

[0191] Twenty-four male SD rats were randomly divided into four groups (6 rats in each group): a control group (Veh), which was intraperitoneally injected with equal volumes of TBS; low-, medium-, and high-dose single-domain antibody groups (SdAb), which were intraperitoneally injected with 2.5, 5, and 10 mg / kg of PDI single-domain antibodies, respectively. 30 minutes after administration, the rats were anesthetized, and after the pain reflex of the rats disappeared, the tails were cut transversely 5 mm from the tip, the first drop of blood was wiped off, and the tails were immersed in 37°C saline. The bleeding was continuously observed: if there was no continuous bleeding within 60 seconds, the hemostasis time was recorded; if bleeding occurred again, the time was continued, and the maximum observation time was 30 minutes (the timeout was counted as 30 minutes). The results are as follows: Figure 19 shown.

[0192] Depend on Figure 19 As can be seen, there was no statistically significant difference in bleeding time between the medium and high-dose groups and the control group (P>0.05), indicating that PDI single-domain antibodies did not increase the risk of bleeding within the therapeutic dose range. Notably, the bleeding time in the low-dose group was significantly shorter than that in the control group (P≤0.05), suggesting that at this dose, single-domain antibodies may exert a procoagulant effect by enhancing platelet activation. This phenomenon suggests that at low concentrations, single-domain antibodies may mediate coagulation regulation by targeting PDI, while at medium and high doses, the pharmacological effects tend to be balanced and do not interfere with the normal physiological process of hemostasis.

[0193] 2) Establishment of thrombus model and thrombus determination:

[0194] The anti-thrombotic effect of PDI single domain antibody was evaluated using FeCl3-induced rat carotid artery thrombosis model. The experiment was performed after the tail-cut bleeding experiment.

[0195] After the tail-cut bleeding experiment, the rat was fixed on a dissecting table and the neck was disinfected with alcohol. The left neck of the rat was dissected with a scalpel and forceps to expose the left common carotid artery. Filter paper, 5 mm x 1 cm, soaked in 35% FeCl3, was wrapped around the left common carotid artery for 15 minutes. The filter paper was removed and, after waiting for another 45 minutes, the thrombus-forming vessel in the rat was cut to a length of 1 cm. The vessel was weighed and recorded as the thrombus weight and photographed. The results are shown below. Figure 20 shown.

[0196] Depend on Figure 20 It can be seen that after the rat carotid artery was cut, the blood vessels in the high-dose group were obviously curled, indicating that the amount of thrombus was small ( Figure 20 A); weighing results show ( Figure 20B) showed a significant reduction in thrombosis in the medium- and high-dose groups (P ≤ 0.05), and the antithrombotic effect increased with increasing dose, demonstrating a clear dose-dependent effect. Combined with previous data from a tail-cut bleeding model (the medium- and high-dose groups did not prolong bleeding time, P > 0.05), this suggests that PDI single-domain antibodies effectively inhibit pathological thrombosis without affecting physiological hemostasis.

[0197] In summary, this example systematically evaluated the therapeutic potential of a PDI single-domain antibody through in vitro and in vivo experiments in a rat carotid artery thrombosis model. In vitro experiments demonstrated that the PDI single-domain antibody screened by the present invention inhibited PDI enzyme activity in a dose-dependent manner and significantly reduced platelet aggregation induced by ADP, AA, and PAF. In in vivo experiments, the PDI single-domain antibody reduced thrombus weight, and the tail-cut bleeding time was no different from that of the control group.

[0198] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A disulfide isomerase single domain antibody, characterized in that The disulfide isomerase single domain antibody comprises a variable region, wherein the variable region comprises CDR1, CDR2 and CDR3; The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the CDR1 to CDR3 are defined according to the IMGT definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Kabat definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Chothia definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and the CDR1 to CDR3 are defined according to the Contact definition scheme.

2. The disulfide isomerase single domain antibody according to claim 1, characterized in that The amino acid sequence of the disulfide isomerase single domain antibody comprises: a1) SEQ ID NO: 14; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 14 and having the same function as the protein shown in SEQ ID NO: 14; or a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% identical to SEQ ID NO: 14 and has the same function as the protein shown in SEQ ID NO:

14.

3. A recombinant protein comprising: the disulfide isomerase single domain antibody according to any one of claims 1 to 2; and Optional tag sequence to facilitate expression and / or purification.

4. A biomaterial related to the disulfide isomerase single domain antibody according to any one of claims 1 to 2, or the recombinant protein according to claim 3, wherein the biomaterial comprises at least one of h1) to h8): h1) a nucleic acid molecule encoding the disulfide isomerase single domain antibody according to any one of claims 1 to 2, or the recombinant protein according to claim 3; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) A transgenic cell line comprising the vector described in h4).

5. A conjugate comprising: at least one of the disulfide isomerase single domain antibody according to any one of claims 1 to 2 and the recombinant protein according to claim 3; and a coupling moiety, the coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein; Preferably, the detectable marker is a fluorescent or luminescent marker; Preferably, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase; Preferably, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188. A solid phase carrier, the surface of which is coupled with the disulfide isomerase single domain antibody according to any one of claims 1 to 2 and / or the recombinant protein according to claim 3.

7. Use of at least one of (1) to (5) in the preparation of a product; (1) The disulfide isomerase single domain antibody according to any one of claims 1 to 2; (2) the recombinant protein according to claim 3; (3) The biomaterial according to claim 4; (4) The conjugate according to claim 5; (5) The solid phase carrier according to claim 6; Preferably, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions i1) to i2): i1) Inhibit platelet aggregation; i2) prevention and / or treatment of thrombotic diseases; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j2): j1) detecting the presence or level of disulfide isomerase protein in the sample; j2) Diagnosis of thrombotic diseases.

8. A product comprising at least one of k1) to k4): k1) The disulfide isomerase single domain antibody according to any one of claims 1 to 2; k2) the recombinant protein according to claim 3; k3) the conjugate according to claim 5; k4) The solid phase carrier according to claim 6.

9. The product according to claim 8, characterized in that: The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions i1) to i2): i1) Inhibit platelet aggregation; i2) prevention and / or treatment of thrombotic diseases; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j2): j1) detecting the presence or level of disulfide isomerase protein in the sample; j2) Diagnosis of thrombotic diseases.

10. A method for preparing the disulfide isomerase according to any one of claims 1 to 2 or the recombinant protein according to claim 3, which is obtained by culturing the transgenic cell line according to claim 4.

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