Monoclonal nano antibody for resisting PLAC1 protein and application of monoclonal nano antibody

Abstract

The invention discloses an anti-PLAC1 protein monoclonal nano antibody and application thereof, and relates to the technical field of nano antibody phage display. The monoclonal nano antibody provided by the invention is PLAC1-1028, PLAC1-1029, PLAC1-1030 or PLAC1-1031, and the monoclonal nano antibody can be used for preparing a monoclonal antibody. The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the PLAC1-1028 are respectively as shown in SEQ ID NO.1-3; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the PLAC1-1029 are respectively as shown in SEQ ID NO. 5 to 7; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the PLAC1-1030 are respectively as shown in SEQ ID NO. 9 to 11; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the PLAC1-1031 are respectively as shown in SEQ ID NO. 13 to 15. The camel source monoclonal nano antibody sequence has high affinity and specific recognition effects on the PLAC1 protein; the method can be widely applied to qualitative / quantitative detection of PLAC1 protein expression and diagnosis and treatment of phase tube tumor diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nano antibody phage display, and in particular to a monoclonal nano antibody against PLAC1 protein and application thereof. Background Art

[0002] PLAC1 (placenta-specific protein-1) is a member of the cancer-testis antigen family (CTAs). PLAC1 protein is lowly expressed or not expressed in normal cells, but can be found in high expression in the placenta, testis and certain malignant tumor cells. In the placenta, PLAC1 is mainly expressed in placental trophoblast cells, which plays a very important role in maintaining the normal growth and development of the placenta and embryo. In a variety of malignant tumor tissues, breast cancer, gastric cancer, cervical cancer, liver cancer, lung cancer, and prostate cancer PLAC1 protein are all highly expressed by more than 10% of the placental expression, and play the role of a cancer-promoting gene in the occurrence and development of tumors. The expression of PLAC1 is associated with the clinical pathological characteristics and prognosis of many tumors. It can be seen that PLAC1 plays an important role under normal physiological and pathological conditions. Research on it can not only help us understand embryonic development and the pathogenesis of certain diseases, but also provide new ideas and potential targets for cancer treatment.

[0003] Phage display technology refers to the technology of fusing foreign proteins or peptides with specific phage capsid proteins and displaying them on the surface of phages. This technology can simulate the process of natural selection, achieve high-throughput screening of target molecule-specific receptors, and perfectly combine the genotype and phenotype of the screened protein molecules. It is often used in the screening of monoclonal antibodies against different target proteins.

[0004] Nanobodies refer to a type of heavy chain single domain antibody. Compared with other antibodies, the light chain is naturally missing and the size is only one tenth of the traditional IgG antibody. Therefore, they have the advantages of high penetration, good stability, and simple humanization. They are widely used in the development of antibody drugs or clinical diagnosis of diseases. Common nanobodies are mainly derived from camels and certain cartilaginous fish.

[0005] If we can screen and develop monoclonal nanoantibodies with high affinity and specific recognition for PLAC1 protein, it will not only provide detection antibodies for the diagnosis of PLAC1, but also be of great significance for the molecular targeted therapy of PLAC1. Summary of the invention

[0006] The present invention provides a monoclonal nanoantibody against PLAC1 protein and its application, and uses phage display technology to construct a corresponding phage display camel-derived nanoantibody library, and screens and obtains two camel-derived monoclonal nanoantibody sequences with high affinity and specific recognition of human PLAC1 protein. The specific implementation is achieved by the following technology.

[0007] In a first aspect of the present invention, a monoclonal nanobody against PLAC1 protein is provided, which is monoclonal nanobody PLAC1-1028, PLAC1-1029, PLAC1-1030 or PLAC1-1031;

[0008] The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1028 are shown as SEQ ID NOs.1-3, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1029 are shown as SEQ ID NOs.5-7, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1030 are shown as SEQ ID NOs.9-11, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1031 are shown as SEQ ID NOs.13-15, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1028 is shown as SEQ ID NO.4; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1029 is shown as SEQ ID NO.8; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1030 is shown as SEQ ID NO.12; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1031 is shown as SEQ ID NO.16.

[0010] Furthermore, the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1028 is shown as SEQ ID NO.17; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1029 is shown as SEQ ID NO.18; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1030 is shown as SEQ ID NO.19; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1031 is shown as SEQ ID NO.20.

[0011] The second aspect of the present invention provides a substance, which comprises any one of the following aspects:

[0012] (1) A nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes any one of the above-mentioned monoclonal nanobodies;

[0013] (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express any of the above-mentioned monoclonal nanobodies;

[0014] (3) An antibody derivative, comprising any one of the above-mentioned monoclonal nanobodies;

[0015] (4) A pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned monoclonal nanobodies, or said antibody derivatives;

[0016] (5) A detection product, comprising any one of the above-mentioned monoclonal nanoantibodies, or the nucleic acid molecule, or the vector, or the antibody derivative.

[0017] The third aspect of the present invention provides an application of the above-mentioned monoclonal nanoantibody against PLAC1 protein, wherein the application includes any one of the following aspects:

[0018] (1) Used for detecting the expression of PLAC1 protein, or detecting the occurrence and development of tumors, or evaluating the efficacy of drugs for treating tumors, for purposes other than disease diagnosis and treatment;

[0019] (2) Products for detecting the expression of PLAC1 protein, or diagnosing tumors, or evaluating the efficacy of drugs for treating tumors;

[0020] (3) Used for preparing drugs for treating tumors.

[0021] Furthermore, the product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

[0022] The tumors detected / treated in the present invention refer to tumor types that are significantly correlated with the expression of PLAC1 protein, including but not limited to breast cancer, gastric cancer, cervical cancer, liver cancer, lung cancer, prostate cancer, etc.

[0023] In fact, the monoclonal nanobody provided by the present invention is not limited to detecting the expression of PLAC1 protein or diagnosing and detecting tumors. In fact, based on the high affinity and specific recognition of the monoclonal nanobody of the present invention to the PLAC1 protein, any other environment and scenario requiring qualitative / quantitative detection of the expression of the PLAC1 protein can use the monoclonal nanobody of the present invention.

[0024] It should also be pointed out that the "not for the purpose of disease diagnosis and treatment" claimed in the use of the monoclonal nanoantibody provided by the present invention specifically refers to the use of the monoclonal nanoantibody of the present invention for qualitative / quantitative detection of PLAC1 protein or related tumor detection under non-clinical conditions such as conventional scientific research or laboratory scenarios. In fact, the monoclonal nanoantibody provided by the present invention can also be used in clinical practice to assist in the detection of PLAC1 protein expression or related tumor diseases.

[0025] In the fourth aspect of the present invention, a camel-derived nanoantibody library is provided for screening any of the above-mentioned monoclonal nanoantibodies; the camel-derived nanoantibody library is obtained by extracting total RNA from natural camel-derived PBMCs cells, performing reverse transcription and PCR amplification to obtain the gene sequence of the antibody variable region VHH, connecting the gene sequence of the antibody variable region VHH to a phage expression vector, and transforming it into competent cells.

[0026] The present invention extracts total RNA from alpaca lymphocytes after immunization with human PLAC1 protein, synthesizes cDNA through a reverse transcription kit, and uses the cDNA as a template to obtain camel-derived nanoantibody variable region gene fragments through two rounds of PCR. The camel-derived nanoantibody variable region gene fragments are connected to the phage display vector pCANTAB 5E by molecular cloning, transformed into the host bacteria TG1 competent state, and successfully constructed a phage display camel-derived nanoantibody library.

[0027] Compared with the prior art, the advantages of the present invention are as follows: the present invention constructs a camel-derived nanoantibody library, takes human PLAC1 protein as the target protein, and finally obtains 4 camel-derived monoclonal nanoantibody sequences with high affinity and specific recognition of human PLAC1 protein through in vitro phage display antibody enrichment and panning and high-throughput ELISA screening, and after ELISA and Western Blot detection; it can be widely used in qualitative / quantitative detection of PLAC1 protein expression, as well as the diagnosis and treatment of related tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of 1% agarose gel electrophoresis in Example 1 are shown in FIG.

[0029] Figure 2 The 1% agarose gel electrophoresis result of the first round PCR product in Example 1 is shown.

[0030] Figure 3 1% agarose gel electrophoresis results of the second round of PCR products in Example 1.

[0031] Figure 4 The electrophoresis results of the four monoclonal nanobodies purified by Protein A resin in Example 3 are shown.

[0032] Figure 5 The Western Blot results of Example 4 are shown. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the implementation cases of the present invention, the term "nucleic acid (molecule)" may be a nucleic acid including coding, or a nucleic acid also including additional coding and / or non-coding sequences. The DNA form of the nucleic acid of the present invention includes but is not limited to cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The nucleic acid encoding the high-affinity monoclonal nanobody or its antigen-binding fragment described in the present invention includes but is not limited to a coding sequence encoding only a mature high-affinity monoclonal nanobody or its antigen-binding fragment structure; a coding sequence encoding a mature high-affinity monoclonal nanobody or its antigen-binding fragment and various additional coding sequences; a coding sequence encoding a mature high-affinity monoclonal nanobody or its antigen-binding fragment structure (and optional additional coding sequences) and non-coding sequences, etc.

[0035] In some embodiments of the present invention, the nucleic acid full-length sequence of the high-affinity monoclonal nanobody or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. Generally, it is cloned into a vector, then transferred into a cell, and then the relevant sequence is isolated from the host cell after proliferation by conventional methods. The biomolecules involved in the present invention (nucleic acids, high-affinity monoclonal nanobodies and antigen-binding fragments thereof, etc.) include biomolecules in isolated form.

[0036] The vectors include viral vectors (such as adenoviral vectors, retroviral vectors, adeno-associated viral vectors) and non-viral vectors (plasmids, transposon vectors).

[0037] In some preferred embodiments, the vector is an expression vector.

[0038] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle that can introduce DNA or RNA sequences into a host cell in a manner that transforms the host and promotes the expression (eg, transcription and translation) of the introduced sequence.

[0039] Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrating plasmids.

[0040] The vector may be a recombinant expression vector or a cloning vector. The vector provided by the present invention (eg, an expression vector) contains the nucleic acid sequence provided by the present invention encoding the antibody, at least one promoter operably connected to the nucleic acid sequence and / or at least one selection marker.

[0041] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses, lambda phage, M13 phage, plasmids, and the like.

[0042] The "transformed cells" provided by the present invention refer to the introduction of "foreign" (i.e., external or extracellular) genes or DNA or RNA sequences into host cells so that the host cells express the introduced genes or sequences to produce substances of interest, usually proteins encoded by the genes or introduced sequences. Host cells that receive and express the introduced DNA or RNA have been "transformed."

[0043] In a specific embodiment of the present invention, the host cell includes but is not limited to mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells and the like.

[0044] The present invention provides an antibody derivative, which comprises the high-affinity monoclonal nanobody provided by the present invention, directly or indirectly connected to a complex formed by a connectable substance.

[0045] The attachable species include a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme.

[0046] In a specific embodiment of the present invention, the connectable substance is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biological toxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL), chemotherapeutic agents (such as cisplatin)) or any form of nanoparticles, etc.

[0047] The term "detectable marker" refers to any part that generates a measurable signal by a change in the optical, electrical or other physical index of the molecular state coupled to the part. Such physical indexes include spectral, photochemical, biochemical, immunochemical, electromagnetic, radiochemical and chemical means, such as but not limited to fluorescence, chemiluminescence and chemiluminescence. When used with respect to a labeled detection agent, a "direct label" is a detectable label attached to the detection agent by any means. When used with respect to a labeled detection agent, an "indirect label" is a detectable label that specifically binds to the detection agent. Therefore, an indirect label includes the following parts: the specific binding partner of the detection agent. Biotin and avidin are examples of such parts that are used, for example, by contacting a biotinylated antibody with a labeled avidin to produce an indirectly labeled antibody.

[0048] The pharmaceutical composition provided by the present invention includes a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient includes a pharmaceutically acceptable solvent, a dispersant, an additive, a plasticizer, etc. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

[0049] In other embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition (e.g., an aqueous solution), or the high affinity monoclonal Nanobody and salt are provided in water without a buffer, or contain an aqueous buffer or other type of solvent (e.g., an organic solvent).

[0050] The term "effective amount" as used herein refers to an amount sufficient to achieve a beneficial or desired result. A "therapeutically effective amount" is an amount that achieves a desired therapeutic effect. This amount may be the same or different from a preventive effective amount, which is the amount required to prevent the onset of a disease or disease symptoms. An effective amount may be administered, applied or dosed once or more. The "therapeutically effective amount" (i.e., effective dose) of a therapeutic compound is determined according to the selected therapeutic compound. For example, the composition may be administered once or more per day, once or more per week, once or more per month, or once or more per year. Certain factors may affect the dosage and timing required for the effective treatment of a subject, including but not limited to the severity of the disease or illness, previous treatment, the general health and / or age of the subject, and other diseases present.

[0051] Example 1: Construction of a camel-derived nanoantibody library that specifically recognizes PLAC1 protein

[0052] 1. Animal immunization: A 2.5-year-old male alpaca was purchased (provided by Qingdao Kangda Aibo Biotechnology Co., Ltd.) and immunized with Recombinant Human PLAC1 Protein and N-His protein (AntibodySystem, catalog number YHN07101). 500 μg of PLAC1 Protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points. Immunization was performed every 2 weeks, and blood was collected from the alpaca 7 days after each immunization. The serum titer was determined by indirect ELISA.

[0053] After 4 rounds of immunization, 80 ml of venous blood was extracted and PBMCs were isolated from camel blood using Tianjin Haoyang's camel peripheral blood mononuclear cell separation kit (Cat. No. LDS1078). The cells were added to 1 ml of trizol solution (Invitvogen, Cat. No. 15596018) and lysed using Thermo's PureLink TM The total RNA of the cells was extracted using RNA extraction kit (Cat. No. 12183020) and detected by 1% agarose gel electrophoresis. Figure 1 shown.

[0054] 2. Reverse transcription to obtain cDNA: The total RNA of natural alpaca PBMCs cells was selected as the template and the cDNA was obtained using Vazyme cDNA was obtained by reverse transcription using III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Cat. No.: R312-02).

[0055] Using cDNA as template, Vazyme Max Super-Fidelity DNA Polymerase (Cat. No.: P505-d3) was used to obtain the gene sequence of the antibody variable region VHH by two rounds of PCR amplification. The two-round PCR system is shown in Tables 1 and 2 below, and the two-round PCR reaction system is shown in Table 3.

[0056] Table 1 1st round PCR reaction system

[0057] 2× Buffer 25μl DNA polymerase 1μl dNTP Mix 1μl cDNA 2μl Forward primer (VHH-F1) 2μl Reverse primer (VHH-R1) 2μl <![CDATA[ddH2O]]> Up to 50μl

[0058] Table 2 Second round of PCR

[0059]

[0060]

[0061] Table 3 PCR reaction conditions are as follows (same for two rounds of PCR):

[0062]

[0063] The results of 1% agarose gel electrophoresis of the first round of PCR products are as follows: Figure 2 As shown in Figure 2, DNA fragments of about 740 bp to 630 bp were recovered. The results of 1% agarose gel electrophoresis of the second round of PCR products were as follows: Figure 3 As shown, a DNA fragment of about 450 bp was recovered.

[0064] 3. The target fragment obtained from the second round of PCR and the vector pCANTAB 5E (Amersham biosciences, phage display system) were digested with SfiI double site according to the restriction system shown in Table 4. The restriction conditions were: 50°C for 5-6 h. According to the ligation system described in Table 5, the target fragment was ligated to the vector pCANTAB 5E under the action of T4 ligase (NEB) at 16°C overnight. The ligation conditions were: 16°C for 15 h; 65°C for 10 min for inactivation.

[0065] Table 4 The restriction enzyme digestion system of vector and DNA target fragment is as follows

[0066] Carrier Purpose fragment DNA 30 μg 5μg Fast Digest sfiI 7μl 2μl 10×Fast Digest Buffer 15μl 5μl <![CDATA[ddH2O]]> To 150μl To 50μl

[0067] Table 5 connection system is as follows:

[0068] T4 DNA Ligase (Thermo) 10μl 10×T4 DNA Ligase buffer 15μl Vector (after SfiI digestion) 2μg VHH fragment (after sfiI digestion) 0.75 μg <![CDATA[ddH2O]]> up to 150μl

[0069] 4. The ligation product was transformed into TG1 electrocompetent cells (Lucigen) by electroporation, plated on 2YT-Amp-Glucose solid culture medium, and cultured at 37°C overnight.

[0070] The formula of 2YT solid culture medium is: 1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) NaCl, dissolved in 1L of distilled water.

[0071] The formula of 2YT-Amp-Glucose medium is as follows: 100 μg / ml ampicillin and 2% (w / v) glucose are added to 2YT solid medium.

[0072] 5. Scrape the colonies on the culture medium, mix them evenly, add glycerol to a final concentration of 15%, and store them at -80°C; inoculate the bacterial solution into 2YT-Amp-Glucose medium and culture at 37°C for 1-2 hours until the OD 600 =0.4-0.6.

[0073] Add helper phage and culture at 37°C for 45min-1h; centrifuge the bacterial solution at 3000-5000rpm, discard the supernatant, resuspend the bacteria with an equal volume of 2YT-Amp-Kan medium, and culture overnight at 30°C; the next day, transfer the supernatant into a new centrifuge tube by centrifugation; add 1 / 4 volume of 5×PEG / NaCl solution, mix thoroughly, place on ice or in a 4°C environment, and let stand for 1-2h; then centrifuge and discard the supernatant, resuspend the precipitate with about PBS, and finally obtain a monoclonal nanoantibody library, which is stored at 4°C or -20°C.

[0074] The formula of the 2YT-Amp-Kan culture medium is as follows: 100 μg / ml of ampicillin and 50 μg / ml of kanamycin are added to the 2YT culture medium.

[0075] Example 2: In vitro enrichment and screening of camelid-derived nanoantibodies (VHH) specific for human PLAC1 protein

[0076] The reagents and materials used in this example are shown in Table 6 below.

[0077] Table 6 Reagents and materials

[0078] Material Part Number brand 96-well plate 42592 Costar Tween 20 P2287 Sigma Tris RES3098T-B7 Sigma Glycine G8200 Solarbio PEG 181986 Sigma PBS C10010500BT Life Casein S12003-100g shyuanye Skim milk 6342932 BD

[0079] 1. Immunotube solid phase panning

[0080] (1) Coating: Coat the immunotubes with 50 μg / ml antigen PLAC1 protein, 1 ml per tube, overnight at 4°C; wash the immunotubes three times with 5 ml PBST.

[0081] (2) Blocking: 5 ml 5% skim milk / PBST, 30°C for 1 h; wash twice with 5 ml PBS.

[0082] (3) Incubation: Add 1 ml to the immunotube, with a total volume of 1-2×10 12 pfu of nanolibrary phage and incubate at 30°C for 1.5h.

[0083] (4) Washing: Wash 6-8 times with 5 ml PBST.

[0084] (5) Elution: Add 1 ml of Gly-HCl (pH = 2.2) to the immunotube to elute the phage, incubate at room temperature with shaking for about 6-8 minutes, and add Tris-HCl (pH = 9.6) to neutralize the solution to pH = 7.0-8.0.

[0085] 2. Determine the titer of phage after elution

[0086] (1) Cultivate E. coli TG1 until OD 600=0.4-0.6; mix 10 μL of diluted eluted phage and 190 μL of E. coli TG1.

[0087] (2) After the mixture was incubated at 37°C for 30 min, it was poured onto 2×YT-A (Amp 100 μg / ml) solid culture medium and cultured at 37°C overnight.

[0088] 3. Amplification of eluted phage

[0089] (1) Directly aspirate 800 μl of the eluted phage solution and add it to 10 ml of the logarithmic phase TG1 bacterial solution. Incubate at 37°C for 30 min and incubate at 220 rpm for 30 min.

[0090] (2) Add the bacterial solution to 30 ml of 2YT-Amp-Glucose medium and culture at 37°C and 220 rpm until OD = 0.4-0.6; add 3×10 11 pfu helper phage, incubate at 37°C for 30 min, and then culture at 220 rpm for 45 min-1 h.

[0091] (3) Centrifuge the bacterial solution at 3000-5000 rpm and discard the supernatant. Resuspend the bacterial cells in an equal volume of 2YT-Amp-Kan medium. Incubate at 30°C, 220 rpm overnight.

[0092] (4) The next day, centrifuge the bacterial solution at 8000 rpm, 4°C, for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5×PEG / NaCl solution, mix thoroughly, and place on ice or at 4°C for 1-2 h. Centrifuge and discard the supernatant, and resuspend the precipitate in about 1 ml of PBS. Centrifuge to precipitate impurities and transfer the supernatant to a new centrifuge tube.

[0093] 4. Test the titer of amplified phage

[0094] Same as step 2.

[0095] 5. Enrichment and panning

[0096] Repeat steps 1 to 4 above to perform the second to third rounds of enrichment and panning. The panning method is shown in Table 7 below.

[0097] Table 7 Enrichment and panning methods

[0098] Number of rounds Coating antigen (μg / mL) Blocking solution Washing times 1 PLAC1(40) 5% skim milk / PBST 7 2 PLAC1(40) 5% skim milk / PBST 7 3 PLAC1(40) 5% skim milk / PBST 8

[0099] The results of in vitro enrichment and panning are shown in Table 8 below.

[0100] Table 8 In vitro enrichment and panning results

[0101]

[0102] 6. Polyclonal phage ELISA detection

[0103] (1) Coating

[0104] Coating of immunoplate: 4 μg / ml antigen PLAC1 protein, 100 μl, overnight at 4°C. Control wells were coated with 100 μl PBS.

[0105] Wash three times with 300 μl PBST.

[0106] (2) Blocking: 300 μl 5% skim milk / PBST, 30°C for 1 h. Wash 2-3 times with 300 μl PBST.

[0107] (3) Incubation

[0108] The phage after each round of amplification was diluted with PBS, and the dilution factor increased by 3 times. The initial concentration was 10 12 pfu / ml. Add 100 μl of diluted amplification phage to each well and incubate at 30°C for 1 h.

[0109] (4) Washing: Wash three times with 300 μl PBST.

[0110] (5) Add secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.

[0111] (6) Washing: Wash three times with 300 μl PBST.

[0112] (7) Color development: Add 100 μl of TMB colorimetric solution and color for 3-8 min in the dark, then add 100 μl of 2M HCl to terminate the reaction. Read the result with an ELISA reader (450 nm-620 nm).

[0113] The results of the polyclonal phage ELISA test are shown in Table 9 below.

[0114] Table 9 Polyclonal phage ELISA test results

[0115]

[0116] 7. Monoclonal phage ELISA screening

[0117] (1) Select an appropriate round number (R4), dilute the eluted phage to an appropriate concentration, infect TG1 in the logarithmic phase, and plate it.

[0118] (2) The next day, 96 single clones were picked from the plate and inoculated into a 96-deep-well plate (600 ul of 2YT-Amp-Glucose medium was added to each well) and cultured at 37°C with shaking at 250 rpm for 2 h until the bacterial solution OD = 0.4-0.6.

[0119] 100 μl of bacterial solution was pipetted into each well of the cell culture plate, and sterile glycerol (final concentration was 20%-25%) was added to each well. After mixing, the mixture was stored at -20°C.

[0120] (3) Add helper phage to the remaining 96-well plate medium. The amount of helper phage added = 0.6 × liquid volume × 5 × 10 8 ×30pfu. Stand at 37℃ for 30min, shake at 250rpm, 37℃ for 45min-1h.

[0121] (4) Centrifuge the 96-well plate at 4000 rpm for 5 min, discard the supernatant, resuspend the bacterial solution in each well with 600 μl of 2YT-Amp-Kan medium, and culture overnight at 30°C and 250 rpm with shaking.

[0122] (5) The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 min and take the supernatant for ELISA experiment.

[0123] (6) Coating: Coat the immunoplate with 4 μg / ml antigen PLAC1 protein, 100 μl, overnight at 4°C. Coat the control wells with 100 μl PBS. Wash three times with 300 μl PBST.

[0124] (7) Blocking: 300 μl 5% skim milk / PBST, 30°C for 1 h; wash 2-3 times with 300 μl PBST.

[0125] (8) Incubation: Add 100 μl of supernatant phage to each well and incubate at 30°C for 1 h.

[0126] (9) Washing: Wash three times with 300 μl PBST.

[0127] (10) Secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.

[0128] (11) Washing: Wash three times with 300 μl PBST.

[0129] (12) Color development: Add 100 μl of TMB colorimetric solution and color for 3-8 min in the dark, then add 100 μl of 2M HCl to terminate the reaction. Read on a microplate reader (450 nm-620 nm)

[0130] The results of ELISA test for monoclonal phage No. 1-48 are shown in Table 10. The results of ELISA test for monoclonal phage No. 49-96 are shown in Table 11.

[0131] Table 10 ELISA test results of monoclonal phage No. 1-48

[0132]

[0133] Table 11 ELISA test results of monoclonal phage No. 49-96

[0134]

[0135] According to the results of Table 10-11 above, OD 600 (Antigen Group)-OD 600 (Control group) greater than 1 was defined as a positive clone; these positive clones were sequenced to exclude erroneous antibody sequences and repeated antibody sequences, and finally 4 high-affinity antibody sequences were obtained, namely high-affinity nanoantibodies that specifically recognize PLAC1 protein, named PLAC1-1028, PLAC1-1029, PLAC1-1030, and PLAC1-1031 respectively.

[0136] After sequencing, the amino acid sequence of the heavy chain complementary determining region CDR1 of the nanoantibody PLAC1-1028 is GFTFDDVA, as shown in SEQ ID NO.1; the amino acid sequence of the heavy chain complementary determining region CDR2 is ISSKGVNA, as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain complementary determining region CDR3 is AVMSKGPRDDAAFGS, as shown in SEQ ID NO.3.

[0137] The heavy chain amino acid sequence of Nanobody PLAC1-1028 is:

[0138] QLQLVESGGGLVQPGGSLRLSCAASGFTFDDVAMAWFRQAPGKEREGVACISSKGVNAFYRDSVKGRFTISSDNAKSTVYLQMNSLKVEDTAVYYCAVMSKGPRDDAAFGSWDKGTQVTVFS; as shown in SEQ ID NO.4.

[0139] After sequencing, the amino acid sequence of the heavy chain complementary determining region CDR1 of the nanoantibody PLAC1-1029 is GITLDNYA, as shown in SEQ ID NO.5; the amino acid sequence of the heavy chain complementary determining region CDR2 is ISQSSGST, as shown in SEQ ID NO.6; the amino acid sequence of the heavy chain complementary determining region CDR3 is AVSSKGCKGDASFGS, as shown in SEQ ID NO.7.

[0140] The heavy chain amino acid sequence of Nanobody PLAC1-1029 is:

[0141] QVQLVESGGGLVQPGGSLRLSCVGSGITLDNYAIGWFRRAPGKEREGVSCISQSSGSTYYADSVKGRFTIDDRDSPRNKNGKNTWYLRMDHLKPEDTAVYYCAVSSKGCKGDASFGSWDKGTQVTVFS; as shown in SEQ ID NO.8.

[0142] After sequencing, the amino acid sequence of the heavy chain complementary determining region CDR1 of the nanobody PLAC1-1030 is GFTLETYA, as shown in SEQ ID NO.9; the amino acid sequence of the heavy chain complementary determining region CDR2 is IDSSAAYY, as shown in SEQ ID NO.10; the amino acid sequence of the heavy chain complementary determining region CDR3 is DQSNSNYGRSRSWDY, as shown in SEQ ID NO.11.

[0143] The heavy chain amino acid sequence of Nanobody PLAC1-1030 is:

[0144] QVQLVESGGGLVQAGESLRLSCAASGFTLETYAIGWFRQAPGKERAGVSCIDSSAAYYADSVKGRFTISRDNAKNTVYLQMSNLKPEDTAVYYCAADQSNSNYGRSRSWDYWGQGTQVTVSS; as shown in SEQ ID NO.12.

[0145] After sequencing, the amino acid sequence of the heavy chain complementary determining region CDR1 of the nanoantibody PLAC1-1031 is GFTWDYYN, as shown in SEQ ID NO.13; the amino acid sequence of the heavy chain complementary determining region CDR2 is ISSNGGDT, as shown in SEQ ID NO.14; the amino acid sequence of the heavy chain complementary determining region CDR3 is AADQRSSNYGSDRRWDY, as shown in SEQ ID NO.15.

[0146] The heavy chain amino acid sequence of Nanobody PLAC1-1031 is:

[0147] QVQLVESGGGLVQPGGSLRLSCTASGFTWDYYNIAWFRQAPGKEREGVSCISSNGGDTYYADSVKGRFTISRDNAKNTVYLQMNILKPEDTAVYYCAADQRSSNYGSDRRWDYWGQGTQVTVSS; as shown in SEQ ID NO.16.

[0148] Based on the amino acid sequences of the above-mentioned high-affinity nanoantibodies PLAC1-1028, PLAC1-1029, PLAC1-1030 and PLAC1-1031, the present invention also provides DNA sequences encoding four high-affinity nanoantibodies. The nucleotide sequence encoding nanoantibody PLAC1-1028 is shown in SEQ ID NO.17; the nucleotide sequence encoding nanoantibody PLAC1-1029 is shown in SEQ ID NO.18; the nucleotide sequence encoding nanoantibody PLAC1-1030 is shown in SEQ ID NO.19; the nucleotide sequence encoding nanoantibody PLAC1-1031 is shown in SEQ ID NO.20.

[0149] Example 3: Recombinant expression of monoclonal nanobodies PLAC1-1028, PLAC1-1029, PLAC1-1030, PLAC1-1031

[0150] The gene sequences of the four nanobodies obtained by screening were subcloned into the pATX2 vector (ATAGENIX), with restriction sites EcoR1 / Not1 and human IgG Fc added to the C-terminus. Xten CHO cells were transfected, and the cells were removed by centrifugation 72 hours after transfection; the culture supernatant was purified using Protein A resin (12% Non-reduced SDS-PAGE), as shown in Figure 4 shown.

[0151] Example 4: Identification of monoclonal nanobodies PLAC1-1028, PLAC1-1029, PLAC1-1030 and PLAC1-1031

[0152] 1. ELISA detection of monoclonal nanoantibodies PLAC1-1028, PLAC1-1029, PLAC1-1030 or PLAC1-1031

[0153] (1) Coating: Coat the immunoplate with 5 μg / ml antigen PLAC1 protein, 100 μl, overnight at 4°C. Coat the control wells with 100 μl PBS. Wash three times with 300 μl PBST.

[0154] (2) Blocking: Block with 300 μl 3% BSA / PBS at 37°C for 1.5 h; wash with 300 μl PBST 2-3 times.

[0155] (3) Incubation: Dilute the monoclonal nanobody with PBS, and increase the dilution multiple by 5 times. The initial concentration is 10 μg / ml. Add 100 μl of the diluted nanobody to each well. Incubate at 37°C for 1 hour.

[0156] (4) Washing: Wash three times with 300 μl PBST.

[0157] (5) Secondary antibody: Add 100 μl of secondary antibody diluent (Goat anti Human (H+L)-HRP, 1:5000) (AntibodySystem, Catalog No. PHB96431) and incubate at 37°C for 30 min.

[0158] (6) Washing: Wash three times with 300 μl PBST.

[0159] (7) Color development: Add 100 μl of TMB colorimetric solution and color for 8 min in the dark, then add 100 μl of 2M HCl to terminate the reaction. Read on a microplate reader (450 nm-620 nm)

[0160] The ELISA test results are shown in Table 12 below.

[0161] Table 12 ELISA test results

[0162]

[0163]

[0164] 2. Monoclonal nanoantibodies PLAC1-1028, PLAC1-1029, PLAC1-1030, PLAC1-1031 ELISA Western Blot detection

[0165] (1) SDS-PAGE electrophoresis: 100 ng PLAC1 protein was subjected to SDS-PAGE gel electrophoresis using a 10% polyacrylamide precast gel (Thermo, catalog number XP00102BOX).

[0166] (2) Membrane transfer: The PLAC1 protein separated by electrophoresis was transferred to a nitrocellulose membrane.

[0167] (3) Blocking: Take out the electro-transferred nitrocellulose membrane, wash it once with TBST, and block it with 5% milk / TBST on a shaker at room temperature for 1 h.

[0168] (4) Washing: Rinse with TBST for 2 min.

[0169] (5) Binding of primary antibody and target protein: Monoclonal nanoantibodies PLAC1-1040 and PLAC1-1042 were diluted with 1% BSA / PBST, incubated with nitrocellulose membrane, and stored in a refrigerator at 4°C overnight.

[0170] (6) Washing: Wash three times with PBST, 10 min each time.

[0171] (7) Incubation of secondary and primary antibodies: Place the treated nitrocellulose membrane in a secondary antibody diluent, Goat anti Human (H+L)-HRP, 1:5000 (Antibody System, Catalog No. PHB96431), diluted in 5% milk / PBST, and incubate on a shaker at room temperature for 1 h.

[0172] (8) Washing: Wash 3 times with PBST, 10 min each time

[0173] (9) Color development: Place the treated nitrocellulose membrane with the front side facing downward in contact with the luminescent reagent. After color development for 1.5-2.0 min, observe the results using a gel imaging system.

[0174] Western Blot results are as follows Figure 5 As shown, it can be seen that the four recombinant antibodies obtained can effectively and specifically recognize PLAC1 protein, producing a single clear immunoblot band.

[0175] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A monoclonal nanobody against PLAC1 protein, characterized in that: It is a monoclonal nanobody PLAC1-1028, PLAC1-1029, PLAC1-1030 or PLAC1-1031; The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1028 are shown as SEQ ID NOs.1-3, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1029 are shown as SEQ ID NOs.5-7, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1030 are shown as SEQ ID NOs.9-11, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody PLAC1-1031 are shown as SEQ ID NOs.13-15, respectively.

2. The monoclonal nanobody against PLAC1 protein according to claim 1, characterized in that The amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1028 is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1029 is shown in SEQ ID NO.8; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1030 is shown in SEQ ID NO.12; the amino acid sequence of the heavy chain of the monoclonal nanoantibody PLAC1-1031 is shown in SEQ ID NO.

16.

3. The monoclonal nanobody against PLAC1 protein according to claim 1, characterized in that The nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1028 is shown in SEQ ID NO.17; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1029 is shown in SEQ ID NO.18; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1030 is shown in SEQ ID NO.19; the nucleotide sequence encoding the monoclonal nanoantibody PLAC1-1031 is shown in SEQ ID NO.

20.

4. A substance, characterized in that The substance includes any one of the following aspects: (1) A nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal Nanobody according to any one of claims 1 to 3; (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the monoclonal nanobody according to any one of claims 1 to 3; (3) An antibody derivative, comprising the monoclonal nanobody according to any one of claims 1 to 3; (4) A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal Nanobody according to any one of claims 1 to 3, or the antibody derivative; (5) A detection product, comprising the monoclonal nanobody according to any one of claims 1 to 3, or the nucleic acid molecule, or the vector, or the antibody derivative.

5. A use of the monoclonal nanobody against PLAC1 protein according to any one of claims 1 to 3, characterized in that: The application includes any one of the following aspects: (1) Used for detecting the expression of PLAC1 protein, or detecting the occurrence and development of tumors, or evaluating the efficacy of drugs for treating tumors, for purposes other than disease diagnosis and treatment; (2) Products for detecting the expression of PLAC1 protein, or diagnosing tumors, or evaluating the efficacy of drugs for treating tumors; (3) Used for preparing drugs for treating tumors.

6. The use according to claim 5, characterized in that: The product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

7. A camel-derived nanoantibody library, characterized in that: Used to screen the monoclonal nanoantibody described in any one of claims 1-3; the camel-derived nanoantibody library is obtained by extracting total RNA from natural camel-derived PBMCs cells, performing reverse transcription and PCR amplification to obtain the gene sequence of the antibody variable region VHH, connecting the gene sequence of the antibody variable region VHH to a phage expression vector, and transforming it into competent cells.

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