A nanobody against CXCL1 and its application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an anti-CXCL1 nanobody and its applications. The nanobody contains a heavy chain variable region targeting CXCL1, composed of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; the amino acid sequence of the nanobody has at least 70% sequence identity with any one of SEQ ID NO. 1-49. The aforementioned nanobody has a small molecular weight, low immunogenicity, better solubility and stability, which is beneficial for basic research and clinical trials. It has potential diagnostic and therapeutic prospects for tumors, autoimmune diseases, and neurological diseases, and has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-CXCL1 nanobody and its application. Background Technology

[0002] CXCL1 (CXC motif chemokine ligand 1) (also known as GRO-α) is a chemokine (CXC subfamily) that primarily binds to the CXCR2 receptor. CXCL1 plays crucial physiological functions, including inducing angiogenesis and neutrophil recruitment, participating in inflammatory responses and immune regulation, and is a key drug target for treating various cancers (such as pancreatic cancer, breast cancer, lung cancer, prostate cancer, liver cancer, and colon cancer), autoimmune diseases (such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and multiple sclerosis), and neurological diseases. Therefore, designing antibodies targeting CXCL1 is of great significance for evaluating human health status, assessing the progression of related diseases, and developing targeted therapies.

[0003] However, current detection and research on CXCL1 mainly focus on CXCL1 inhibitors or monoclonal and polyclonal antibodies. For example, Reparixin is a non-competitive allosteric inhibitor of chemokine receptors CXCR1 and CXCR2 activation; Corydalmine alleviates Vincristine-induced neuropathic pain by inhibiting the NF-κB-dependent CXCL1 / CXCR2 signaling pathway; NTC-001 is a humanized mouse anti-human CXCL1 antibody that significantly inhibits tube formation in human umbilical vein endothelial cells (HUVECs). Existing small-molecule inhibitors suffer from poor targeting and toxic side effects, while full-length antibodies are effective but have a slow onset of action and are not easily able to penetrate deep tissues. Their application in current detection and related disease research and treatment still falls short of requirements, and their specificity and affinity need further improvement. Therefore, the development of novel antibodies with good specificity and affinity is particularly necessary. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides an anti-CXCL1 nanobody and its applications. This invention successfully prepared an anti-CXCL1 nanobody with high affinity, stability, and specificity, which can be used for the detection and analysis of CXCL1 and for the diagnosis and treatment of CXCL1 protein-related diseases, especially those related to abnormally high CXCL1 expression. This invention is based on the above research findings.

[0005] In a first aspect, the present invention provides an anti-CXCL1 nanobody, said nanobody comprising a heavy chain variable region targeting CXCL1, composed of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Preferably, the CDR region of said nanobody may be composed of a combination of three CDR regions of any of the sequences in SEQ ID NO. 1-49 shown in Table 1.

[0006] Table 1. Antibody amino acid sequence structures as shown in SEQ ID NO.1-49

[0007] Preferably, the amino acid sequence of the nanobody has at least 70% sequence identity with any one of SEQ ID NO. 1-49; more preferably, it has at least 80% sequence identity with any one of SEQ ID NO. 1-49; even more preferably, it has at least 90% sequence identity with any one of SEQ ID NO. 1-49; even more preferably, it has at least 95% sequence identity with any one of SEQ ID NO. 1-49; most preferably, as shown in any one of SEQ ID NO. 1-49.

[0008] In a second aspect, the present invention provides an isolated nucleic acid molecule capable of encoding the aforementioned anti-CXCL1 nanobody.

[0009] The nucleic acid sequence encoding antibody Y1-1A is as follows: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-1C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATAGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-1D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGCATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCCGCCTCTGGACGCCCCATCAATAACTATGCCATGACCTGGTTCCGCCGGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAATAATTAATAACAGTGGTACGATCACTTACTATACAGAGCGCGTGAAGGGTCGATTCACCATCTCCAGAGACGACGACCTGAGCACGGTGTTTCTGCAAATGAACGACCTGAAACCGGAGGACACGGCCGTTTATTACTGCGCAGCCGGAGTGCGGAATTATCGTCCCCTTAGTACCTACACCCCGCGTGACTTCGTTTTCTGGGGTCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-1H is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCTGCTATTAGCTGGAGTGGTGGTACCACACACTATGCAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCCTCCCCCCGCACAAGGCATACTATGCCGGTACTTACTACTCTCCTTCAGAGTATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-2A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATGGCAGCGATTGCTAGGAGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTTAGTCCAACTCCCTACTACCGCGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-2B is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATAGCAGCGATTGCTAGGGGTGGTGGTAGGACAGACTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAGCGATTATGCGTGGGCTTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-2C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGCGAGAGTGGTGGATTTACTCACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGACTTCAAGCCTACATATTATAGCGGTAGGCACAACCCGACCGAAGCTGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-2E is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-3E is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTCGGTCGCTATGCCATGGGCTGGTTCCGCCAGAGTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTAGTTGGAGTGGTGATAACACGCAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGATCCCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATTCATCTCCGACGCCCTTTTGGACAGTAGGTGGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-3F is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACTTCCCTTCAGTAGCTACGCCATGGGCTGGTTCCGCCAGGCTCTAGGGAAGGAGCGTGAGTTTGTCGCAGCGATTACTTGGAGTGGTGGTAGGACAGAGTTCGCAGACTCCGTGCAGGGCCGAGGCACCATCTCCAGAGACAACGACAAGAGCACGGTGTCTCTAACAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCAGCGGATCAGAGTCCCGTCCCCTACTACGAGGGAGAGGGCTGGGAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-3G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCATGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-3H is: GATGTGCAGCTGCAGGAGTCTGGGAGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATAGCAGCGATTGCTAGGGGTGGTGGTAGGACAGACTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAGCGATTATGCGTGGGCTTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-4A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGACTGGGGAGTCCCTGAGACTCTCCTGCGCAGCCTCTGGACGCACCAGTAGCAGTCTAGTTTTGGGCTGGTTCCGCCAGACTCCAGGAAAGGAGCGTGAATTTGTTGCGGCTATTGGTTGGAAGACGACAGACTCTGTAACCGGCGGTCCCTGGCAATACTATGCCGACTCCGTGAGGGGCCGGTTCACCGTCTCCGGAGACAACGCCAAGACCACGGTGTATCTGCAAATGAACGGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCGCGGCGCGGTATTTGGAATTACGCGTCGGGGACTTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-4G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGCATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCCGCCTCTGGACGCCCCATCAATAACTATGCCATGACCTGGTTCCGCCGGGCCCCAGGGAAGGAGCGTGAGTTTGTAGCAATAATTAATAACAGTGGTACGATCACTTACTATACAGAGCGCGTGAAGGGTCGATTCACCATCTCCAGAGACAACGACCTGAGCACGGTGTTTCTGCAAATGAACGACCTGAAACCGGAGGACACGGCCGTTTATTACTGCGCAGCCGGAGTGCGGAATTATCGTCCCCTTAGTACCTACACCCCGCGTGACTTCGTTTTCTGGGGTCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-5B is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGACTCTCTGACACTCTCCTGCGCGGACTCTGGACGCGCCACGGATTCATATTCCGTGGGCTGGTTCCGCCAGGCTCCAGGAAAGGACCGTGAGTTTGTGGCAGTCATTAGCTGGATTGCAGGTATCACATATTATGCAGAATCCATTCAGGGCCGATTCGTCGTCTCCAGAGACAACGCCAAGAACACGGTGTCTCTACAAATGAACAGCCTAAAACCCGAGGACACGGCCGTTTATTACTGTGCAGCCCAACCTAGGAAAACCTGGTACCGGGCGGCGGAAGAGGATCGCTATTCGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-6A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGGTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTTCGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAACTGGAGTGGTACTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCGCCAAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTAGCGGATTGGGGTACGTAACCCCCGGCGAATATGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-6C is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGACCGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGATGATTATGCCATCGGCTGGTTCCGCCAGGCCCCGGGGAGGGGGCGTCAGGGGGTCGCATGCATTAGTGCTGCTGATGGTAGTATATACTATGGACACTCCGTGAAGGGCCGATTCACCATGTCCAGTGACAGCGCCAACAACATGTTCTATCTAGAAATGAATATGCTACAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGATCTAATTTGCCCTTTGGGATTGGGACACGAATTGAGATATTCGCATGACTACTCGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-6D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCCGCCTCTGGACGCACCATCAGTAGTACTGCCATGGCATGGTTCCGCCAGGCTCCAGGGAAGGGACGTGAATTTGTAGCCGCTATTAGGTGGAGTGATGGAGACACATCTTATACAAGTAACGTGCAGGACCGATTCATTATCTCCAGAGACAACGCCAACAGCACCATGTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGCACGCCCAACCGCTAGTACTAGGCTCGTCTACATTAGGGACTATGAGTATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCCATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTCGGTCGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTAGTTGGAGTGGTGGTAACACGGAGTATGCAGACTCCGTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACACGGTGTATCTACGTATGAACAACCTGATCCCTGAGGACTCGGCCGTTTATTACTGTGCAGCAGATTCATCCCCGACGCCCTTTTGGGTAGTAGGTAGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCATCTTCAGTAGCGCTGCCATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTGGTAGCACGTATACTCTGGAGCGGTGGTAGCACATACTATGAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTATGTGCAGTAGCAAAGTCCTACGCGCCGTTTCGCGATGTTTCTTCTTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCCCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAACTGGAGTGATGGTAAGTCATTCTATGCAGACTCCGTGAAGGGCCGAGTCACCGTCTCCAGAGACAACGCCAAGAACACGGGGTATCTGCAAATGAACAGCTTGAAACCTGAGGATACGGCCGTTTATTACTGTGCAGCTGATCGGGAATTATACTATAGTGGTAGTTACTACCGCGCCGAGGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-8G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACTTCCCTTCAGTAACTACGCCATGGGCTGGTTCCGCCAGGCTCTAGGGAAGGAGCGTGAGTTTGTCGCAGCGATTACTTGGAGTGGTGGTAGGACAGAGTTCGCAGACTCCGTGCAGGGCCGAGGCACCATCTCCAGAGACAACGGCAAGAGCACGGTGTCTCTAACAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCAGCGGATCAGAGTCCCGTCCCCTACTACGAGGAAGAGGGCTGGGAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-8H is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATAGCAGCGATTGCTAGGGGTGGTGGTAGGACAGACTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAGCGATTATGCGTGGGCTTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-9D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTCGGTCGCTATGCCATGGGCTGGTTCCGCCAGAGTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTCGTTGGAGTGGTGATAACACGCAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGATCCCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATTCATCTCCGACGCCCTTTTGGACAGTAGGTGGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-9E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTACCGATACCATGGCCTGGTACCGCCAGGCCCCAGGGAAGCAGCGCGCATTGGTCGCAACTATTGCAAGTGATAGTAGAACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGCCGTCTATTACTGCAAAGGCATAATTAGAGATAGCTGGTACGCCCCTCTCGCGGACTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-10F is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGCGTCTCCTGTGCAGCCTCTGGTCTTACCTTCAGTAACTACACCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCAGTTATTGGTAGGAGTGGTCGTTACTCAAGGTATGCAGACTCCGTGAAGGGCCGATTCATCATCTCCAGAGATAACACCAAGAGCAACGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACTGGCGTTTATTACTGTGCAGCCGCCCTGATATTCAGGTTAGATCCAAAGCCTGACTATTGGGGCCAGGGAACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-10G is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCATTTTCAGTACCTATGGCGTGGGCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTGCTAGGAATGGTGGAAACATACAGTATGGGGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTTAGTCCAACTCCCTACTATAGGGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-10H is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATATAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-11A is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGTCATCTCTGAGACTCTCCTGCGCAGCCTCTTCTGTAGCCTCTGAACGCACCCTCAACGACTACACCGTGGGCTGGTTCCGCCAGGGTCCAGGGACGGAGCGTGAATTTGTAGCAGCGGTTAGTTGGCTCCGTGAGAATACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAACCTGAAACGTGAGGACACGGCCGTTTATTATTGCGCAACCCGGACGGGAAGTCTTACTTCGCGGACGGAATATGAGTACGACTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-11E is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGAGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAACTATTAATAGTGGTGGTGGTATCACAACCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTGTATTACTGTACAAAAGCGGGGGCGAGCGACCCCCCCCCGGGGCCAGGGGACCAGGTCACCGTCTCCAGCGG; The nucleic acid sequence encoding antibody Y1-12A is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTAGGTCGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTAGTTGGAGTGGTGGTAACACGGAGTATGCAGACTCCGTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACACGGTGTATCTACGTATGAACAACCTGATCCCTGAGGACTCGGCCGTTTATTACTGTGCAGCAGATTCATCCCCGACGCCCTTTTGGGTAGTAGGTAGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid sequence encoding antibody Y1-12E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGTTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-12F is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGGAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-12G is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGTTATTAGCTGGAGTGGTAATTTCACATACTATGCTGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTGCTGTGCAGCAGACTTCGAGCCAACATACTTTAGTGGTAGACACAGTCCGACCGAAGCTGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-1G is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGATGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTACACGCACCTTTAGTAGATATGTTATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGACTTTGTAGCAGCTATTAGCAGGACTGCTAATGCAGTCCATACAGACTCCGTGAAGGGTCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGGTGTCGGGGGATACTATGGTAGTATTGAGGGGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-2C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGGGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-2D is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGTAGCATCAGTCGTCAAGCCATGGGCTGGTTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAACGGCTATGAACTACAATGGTGAAAGTATGGTCTATGCAGCATTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATACGGTGTATCTACAAATGGACAGCCTGAAACCTGAAGACACGGCCGTATATTATTGTGCGGCAGGTCACTACGGATTGAGGTACGACATGTCGGCCGTGAGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-3F is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATGGCAGCGATTGCTAGGAGTGGTGGTAGCACATTCTATGGAGGCTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTTAGTCCAACTCCCTACTACCGCGATTATGCGTGGGCTTCCTGGGGTCAGGGGACCCAGGTCACCGTCTCCAGCGG The nucleic acid molecule sequence encoding antibody Y2-4C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTGGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-6E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAGCACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-6G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGAGTTTATGGCAGCGATTGCTAGGAGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGTACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTTAGTCCAACTCCCTACTACCGCGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-9A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTGTAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-9B is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTTCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-10A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGTTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-11A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGTAGCATCAATCGTCAAGCCATGGGCTGGTTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAACGGCTATGAACTGGAATGGTGAAAGTATGGTCTATGCAGCATTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATACGGTGTATCTACAAATGGACAGCCTGATTCCTGAAGACACGGGCGTTTATTATTGTGCGGCAGGTCACTACGGATTGAGGTACGACATGTCGGCCGTGAGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-11H is: GATGTGCAGCTGCAGGAGTCTGGAGGAGCATTGGTGCAGGCTGGGGCGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTGCATATGCCAAGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCGATTGACTGGAGTGGCAGTCGCACTGAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAACACCAAGAACACGGTGTACCTCCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATTCTTCCCCGACGCCCTTCTTCCTTGTCTCTGGTTGGCGCGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-12D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAAAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCT ATGGAGACTCCGTGCTGGGCCGATTCACCATTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y2-12F is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTTGAGACTCTCCTGTGTAGCCTCCGGATTCACCTTCGGTAATTGGATGCATTGGGTCCGTCAGGCTCCGGGGAAGGGGCTCGAGTGGATCTCGGGAATAAATGAGGCGAATAGTGA AACATGGTATGGAGACGCCGTACAGGGCCGATTCACCATCTCCAGAGACAACTCCAGGAACACGCTACATCTGCAATTAATGAGTCTGAAGGCTGACGATTCGGCCAAGTATTACTGTGTGAGAGACCGCGGGAATTACGGTGGATATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGCGG.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecules.

[0011] The recombinant expression vector is obtained by ligating the nucleic acid molecule to an expression vector; wherein the expression vector may be selected from: DNA, RNA, viral vectors, plasmids, bacteriophages, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, transposons, other gene transfer systems, or combinations thereof. A fourth aspect of the invention provides a host cell comprising the above-mentioned recombinant expression vector, or comprising the above-mentioned nucleic acid molecule integrated into the host cell genome; or, the host cell expresses the above-mentioned anti-CXCL1 nanobody.

[0012] Furthermore, the host cells include cells derived from prokaryotes and eukaryotes. Among them, prokaryotes are preferably Escherichia coli, and eukaryotes include yeast and mammalian cells, including CHO, NSO, HEK293, PERC6, etc., without specific limitations.

[0013] A fifth aspect of the present invention provides an immunoconjugate comprising the anti-CXCL1 nanobody described in the present invention, and a conjugation portion, wherein the conjugation portion is a detectable marker, drug, toxin, cytokine, viral capsid protein, or virus-like particle, etc.

[0014] The detectable markers include radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or enzymes.

[0015] The drug may be a cytotoxic drug, such as an anti-tubulin drug, DNA minor groove binder, DNA replication inhibitor, alkylating agent, antibiotic, folic acid antagonist, antimetabolite, chemotherapy sensitizer, topoisomerase inhibitor, vinca alkaloid, etc.

[0016] The toxins mentioned may be chlortetracycline, levofloxacin, ricin, ethidium bromide, mitomycin, diphtheria toxin, absinthecin, white tree toxin, metoprolol, localized aspergillin, phenolmycin, jatropha toxin, croton toxin, chalcogenide, glucocorticoids, etc., without specific limitations.

[0017] The immunoconjugate contains multivalent (e.g., bivalent) anti-CXCL1 nanobodies as described in this invention. The multivalent meaning refers to the presence of multiple repeating anti-CXCL1 nanobodies as described in this invention within the amino acid sequence of the immunoconjugate.

[0018] A sixth aspect of the invention provides a conjugate comprising the above-described anti-CXCL1 nanobody and a conjugation portion; preferably, the conjugation portion is selected from protein tags, detectable markers, therapeutic agents, or other bioactive peptides.

[0019] The protein tag includes a purification tag; the detectable label includes any one of an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin; and the therapeutic agent includes any one of an antitumor drug, an anti-inflammatory drug, or an immunosuppressant.

[0020] A seventh aspect of the present invention provides a detection kit comprising the above-described anti-CXCL1 nanobody, and / or the above-described immunoconjugate, and / or the above-described conjugate; the kit can be used to detect the presence or level of CXCL1 in a sample.

[0021] The kit can be used for non-invasive detection of CXCL2 expression in test subjects.

[0022] An eighth aspect of the present invention provides a pharmaceutical composition comprising the above-described composition, the above-described immunoconjugate, or the above-described conjugate.

[0023] Furthermore, it also includes pharmaceutically acceptable carriers and / or excipients. Moreover, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0024] The non-pharmaceutical active ingredients, such as carriers and / or excipients, that may be included are well known in the art and can be determined by those skilled in the art to meet clinical standards.

[0025] Furthermore, the pharmaceutical compositions of the present invention can be administered into the body in known ways. For example, they can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, administration can be made via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0026] The pharmaceutical composition can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees. Among these, humans are the most preferred.

[0027] A ninth aspect of the present invention provides the use of the above-described anti-CXCL1 nanobody, the above-described immunoconjugate, the above-described conjugate, the above-described kit, or the above-described pharmaceutical composition in any one or more of the following: (a1) To prepare products for the detection, monitoring, diagnosis and / or prognosis of tumors, autoimmune diseases or neurological diseases; (a2) To prepare products for the prevention and / or treatment of tumors, autoimmune diseases, or neurological diseases; The tumor, autoimmune disease, or neurological disease is specifically characterized by abnormally high expression of CXCL1.

[0028] The tumors include pancreatic cancer, breast cancer, lung cancer, prostate cancer, liver cancer, and colon cancer, etc.; the autoimmune diseases include rheumatoid arthritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, and multiple sclerosis, etc.; and the neurological diseases include Alzheimer's disease, etc.

[0029] In a tenth aspect of the present invention, the application of the above-described anti-CXCL1 nanobody in the preparation of fusion proteins is provided.

[0030] Furthermore, the fusion protein comprises a therapeutic protein and the aforementioned anti-CXCL1 nanobody.

[0031] This invention also discloses a method for screening the above-mentioned anti-CXCL1 nanobodies, which specifically includes the following steps: (1) Immunize alpacas with recombinant CXCL1 protein; (2) Isolate peripheral blood lymphocytes from alpacas, extract total RNA, and reverse transcribe to synthesize cDNA; (3) Two rounds of PCR amplification of the target fragment were performed using primers to construct phage particles, which were then transformed into phages to construct a phage library. Positive clones were eliminated in multiple rounds. The primers are shown below: First-round primers: CALL 001: GTCCTGGCTGCTCTTCTACAAGG; CALL 002: GGTACGTGCTGTTGAACTGTTCC; Second-round primers: VHH-For: GACTAGTCACTAGTGATGTGCAGCTGCAGG; VHH-Back: CGAGCTCGGAGCTCTGAGCGG.

[0032] (4) Positive clones were constructed into expression vectors, and expression and purification were induced using a prokaryotic expression system. The affinity, thermal stability and biological activity with CXCL1 were measured, thus completing the screening of anti-CXCL1 nanobodies.

[0033] This invention provides the amino acid sequence of a highly specific and high-affinity CXCL1-binding nanobody, as well as its construction method and expression and purification strategy. Those skilled in the art can refer to the content of this document to appropriately modify the process parameters.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention uses recombinant CXCL1 to immunize alpacas, establishes a nanobody phage library, and screens out nanobody sequences that can bind to CXCL1 with high affinity. These nanobody sequences have small molecular weight, low immunogenicity, better solubility and stability, which are beneficial for basic research and clinical trials. They also have potential diagnostic and therapeutic prospects for tumors, autoimmune diseases and neurological diseases, and have good practical application value. Attached Figure Description

[0035] Figure 1 The above are the agarose gel electrophoresis results of total RNA from alpaca PBMCs in this embodiment of the invention. Figure 2 The results of random colony PCR agarose gel electrophoresis of bacterial libraries in this embodiment of the invention are shown. Figure 3A The above are the SDS-PAGE electrophoresis results of some TransB expression purification antibodies in the embodiments of the present invention; Figure 3B The above are the SDS-PAGE electrophoresis results of some TransB expression purification antibodies in the embodiments of the present invention; Figure 4A This is a schematic diagram showing the SPR determination results of the nanobody affinity constant in an embodiment of the present invention; Figure 4B This is a schematic diagram showing the SPR determination results of the nanobody affinity constant in an embodiment of the present invention; Figure 4C This is a schematic diagram showing the SPR determination results of the nanobody affinity constant in an embodiment of the present invention; Figure 4D This is a schematic diagram showing the SPR determination results of the nanobody affinity constant in an embodiment of the present invention; Figure 5 This invention illustrates the effect of nanobodies on HCT116 cell clone formation in an embodiment of the present invention. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0038] Example 1. Immunotherapy, library preparation, and screening of Anti-CXCL1 nanobodies The recombinant CXCL1 protein was selected as the antigen for immunizing alpacas. The antigen was injected bilaterally near the cervical lymph nodes of the alpacas, with immunization occurring every fourteen days. After each immunization, 5 mL of peripheral blood was drawn from the alpacas, and plasma was separated for antibody titer determination. A total of five immunizations were performed, and the plasma titer reached 10. 4 level.

[0039] 1.1 RNA extraction and reverse transcription (1) Transfer peripheral blood lymphocytes preserved with Trizol to a 1.5 mL centrifuge tube and add 1 / 5 volume of chloroform and mix well; (2) After standing at room temperature for 5 minutes, centrifuge at 12000g at 4℃ for 15 minutes; carefully transfer the supernatant after centrifugation to a new centrifuge tube; add an equal volume of isopropanol to the new centrifuge tube; after standing at room temperature for 10 minutes, centrifuge at 12000g at 4℃ for 10 minutes; (3) Wash each tube of precipitate with 1 mL of 75% ethanol, centrifuge at 7500g for 5 minutes, remove the ethanol, dry, dissolve the precipitate in an appropriate amount of RNase-free water, combine all samples, and that is the total RNA extracted; (4) Use the Takara reverse transcription kit to reverse transcribe the obtained total RNA. Divide the above total RNA sample into two parts, one part using Oligo dT Primer in the kit as primer, and the other part using Random 6-mers in the kit as primer, and reverse transcribe the total RNA obtained in the previous step into cDNA according to the reverse transcription kit instructions, and save them into two centrifuge tubes respectively.

[0040] 1.2 PCR amplification First round of PCR amplification: (1) The first round of PCR reaction was performed using cDNA as a template. The primers for the first round of amplification were: CALL 001: GTCCTGGCTGCTCTTCTACAAGG; CALL 002: GGTACGTGCTGTTGAACTGTTCC. PCR amplification was performed using Taq DNA Polymerase Hot Start enzyme. The reaction system consisted of 0.5-4 μL cDNA, 2 μL / 2 μL CALL 001 / CALL 002, 4 μL dNTP Mix, 5 μL 10×ExTaq Buffer, 0.25 μL HS Ex Taq, and up to 50 μL ddH2O. The reaction conditions were: Step 1, 98℃, 3 min; Step 2, 94℃, 50 s, 55℃, 30 s, 72℃, 40 s + 2 s / cycle; Return to Step 2, 23 cycles; Step 3, 72℃, 5 min; Step 4, 4℃, forever. (2) After the reaction, take 20 μL of PCR product for 1% agarose gel electrophoresis. Finally, select the template amount with a single target band and a fragment size of 600 bp in the electrophoresis result as the best template amount. Perform PCR reaction on all cDNAs according to this template amount and under the same conditions. (3) Perform 1% agarose gel electrophoresis on all PCR products and cut the gel to recover the target fragment size of about 600 bp. (4) Collect all the purified and recovered products of the first round of PCR into a centrifuge tube, which is the first round of PCR amplification product. Store at -20℃.

[0041] Second round of PCR amplification: (1) The first-round PCR amplification product was used as a template for the second-round PCR reaction. The primers for the second-round amplification were as follows: VHH-For: GACTAGTCACTAGTGATGTGCAGCTGCAGG; VHH-Back: CGAGCTCGGAGCTCTGAGCGG. In order to determine the optimal amount of template used, 0.3 μL, 0.5 μL, 1 μL, 2 μL and 3 μL were used as templates respectively. The reaction system was configured as follows: 0.3-3 μL of the recovered product from the first round PCR, 2 μL / 2 μL of VHH-For / VHH-Back, 4 μL of dNTP Mix, 5 μL of 10×ExTaqBuffer, 0.25 μL of HS Ex Taq, and ddH2O up to 50 μL. The reaction conditions are as follows: Step 1, 98℃, 3min; Step 2, 94℃, 50s, 55℃, 30s, 72℃, 40s; switch to Step 2, 11 cycles; Step 3, 72℃, 5min; Step 4, 4℃, forever. (2) After the reaction, take 20μL of PCR product for 1% agarose gel electrophoresis. Finally, select the template amount with a single target band and a fragment size of about 400bp in the electrophoresis result as the best template amount. Take about 1 / 5 of the volume of the obtained first round PCR amplification product, a total of 288 reactions, according to this template amount and using 1.2 (1) After performing PCR reaction under the same conditions, use a universal DNA purification and recovery kit to purify DNA in the PCR reaction solution; (3) Collect the recovered product into a centrifuge tube, which is the second round PCR amplification product. At the same time, take 2μL and use a nucleic acid concentration meter to detect and record the concentration of the recovered product. Store the remaining product at -20℃.

[0042] 1.3 Enzyme digestion and ligation Vector and PCR product digestion: (1) Using pComb3XSS as the phage plasmid vector, restriction endonuclease was used. Spe I, Sac I. Digest 12 μg of pComb3XSS vector and 4 μg of the second-round PCR amplification product with enzymes respectively, and incubate at 37℃ for 4 h; (2) Purify the pComb3XSS vector and the second-round PCR amplification product using a DNA recovery and purification kit, and store at 4℃. Ligate the vector and fragment, and the reaction system is as follows: PCR product 1.6 μg, vector (pComb3XSS) 4 μg, T4 ligase 30 μL, 10×T4 reaction buffer 200 μL, ddH2O Up to 2000 μL; (3) Incubate the ligation reaction at 4°C overnight (about 16 h); (4) Purify the ligation reaction solution using a universal DNA purification and recovery kit, detect the concentration of the recovered product, and store at 4°C.

[0043] 1.4 Construction of bacterial and bacteriophage libraries 1.4.1 Verify the conversion rate of the ligation product (1) Take a 50 μL TG1 competent cells and place it on ice for 5-10 min to thaw; (2) Add 100 ng of ligation product and transfer it to a pre-cooled electroporation cuvette with a spacing of 1 mm. Set the parameters in the electroporator to 1800 V and 1 mm, and then click the button to click transformation; (3) After the electroporation is completed, immediately add 1 ml of SOC culture medium preheated at 37℃, mix well, and then shake the bacteria at 37℃ and 220 rpm for 1 h to recover; (4) Take 100 μL of the recovered bacterial solution for 10-fold serial dilution and plate it. Calculate the number of transformed colonies that can be obtained for each reaction based on the dilution factor and the number of single colonies. This is the transformation efficiency of the ligation product; (5) At the same time, randomly select 48 single clones for colony PCR. A single band of about 400 bp in the PCR product is considered a positive clone. The positive rate of single clones is estimated from this.

[0044] 1.4.2 Construction of bacterial libraries (1) Take 24 100ng ligation systems and perform electroporation using 24 tubes of competent cells as described above; (2) After recovery at 37℃ for 1 hour, take 100μL of the solution and perform a 10-gradient dilution, then plate it and incubate overnight at 37℃; (3) Collect all remaining bacterial solutions and spread them evenly into 5 245mm square culture plates (2×YT containing 100μg / ml Amp, 2% glucose, and 2% agarose), and incubate overnight at 37℃; (4) Calculate the number of transformed colonies that can be obtained from all reactions based on the dilution factor and the number of single colonies, which is the library capacity of the bacterial library; (5) At the same time, randomly select 48 single clones from the gradient dilution plates for colony PCR to verify the cloning positivity rate of the bacterial library; (6) Scrape the colonies from the overnight cultured 245mm square culture plate with 2×YT liquid medium, place them in a 50ml centrifuge tube, measure their OD600 value, add a final concentration of 20% glycerol, and store at -80℃.

[0045] 1.4.3 Preparation of Phage Library (1) Calculate the volume of bacterial library to be added to 100ml 2×YT liquid medium based on the OD600 of the bacterial library. Inoculate the bacterial library into 100mL 2×YT liquid medium (containing 100μg / ml Amp) according to the calculation results, and incubate at 37℃ and 250rpm until the OD600 is 0.5-0.55; (2) Add helper phages at a ratio of 1:20 (number of bacteria: number of phages) according to the helper phage titer, and incubate at 37℃ and 250rpm for 30-60min; (3) Add a final concentration of 50 μg / ml Kana, and incubate overnight at 30℃ and 250rpm. (4) Centrifuge the overnight cultured bacterial solution at 4℃ and 8000rpm for 10min, and then transfer the supernatant after centrifugation to a new 50ml centrifuge tube; (5) Add 1 / 4 of the pre-cooled PEG / NaCl stock solution, mix well, and incubate on ice for 30min; (6) Centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, and drain by inverting for 2min; (7) Add 5mL of PBS to resuspend in a new centrifuge tube, and centrifuge at 4℃ and 8000rpm for 10min; (8) After centrifugation, transfer the supernatant to a new centrifuge tube, add 1 / 4 of the pre-cooled PEG / NaCl stock solution again, mix well, and incubate on ice for 10min; (9) Centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, resuspend in 1mL of PBS, centrifuge at 8000rpm for 10min, transfer the supernatant to a new centrifuge tube, and store at -80℃. This is the purified phage library.

[0046] 1.5 Immune Screening 1.5.1 First Round of Screening (1) Remove the screening antigen from the -80℃ freezer and thaw it on ice; (2) Coat the screening antigen onto the immunotubes (50µg / tube, PBS coating solution, 2ml / tube), and simultaneously coat the milk control in parallel. Rotate the coating slowly at 4°C overnight; (3) Discard the liquid in the overnight coated immunotubes, add 2ml PBS buffer and wash the immunotubes 3 times at room temperature, rotating for 5min each time; (4) Add 2ml blocking solution (3% skim milk powder) and rotate to block at room temperature for 2h; (5) Discard the liquid in the blocked immunotubes, add 2ml PBST buffer and wash the immunotubes 3 times at room temperature, rotating for 5min each time; (6) Discard the washing solution in the immunotubes, add 2ml PBS, calculate and add the prepared phage library according to the following formula as the first round of screening input phage library, and incubate at room temperature for 1h:

[0047] Where V is the volume of phage added (unit μL), and Tlibrary is the phage titer; (7) Discard the liquid in the immunotube, add 2ml PBST buffer and wash the immunotube 20 times at room temperature, rotating for 5min each time; (8) Discard the liquid in the immunotube, remove as much residual liquid as possible, add 1ml 0.25mg / ml Trypsin solution, and elute by rotating at room temperature for 30min; (9) Add 10μL 10% AEBSF to stop elution, and transfer the solution in the immunotube to a new 1.5ml centrifuge tube, which is the first round of phage screening elution solution.

[0048] 1.5.2 First-round phage eluent titer detection (1) Streak a single colony of TG1 strain stored at -80℃ on 2×YT solid medium and incubate overnight at 37℃ (stored at 4℃ for one week). Pick a single colony from the single colony plate and transfer it to 5 ml of 2×YT medium and incubate overnight at 37℃; (2) Transfer 500 μL of the overnight culture to 5 ml of 2×YT liquid medium and incubate at 37℃ and 220 rpm for about 45 min-60 min until the OD600 is 0.5-0.55; (3) Take 10 μL of the first round of phage elution buffer and incubate in 1 (3) Dilute 10-fold in 0.5 ml centrifuge tubes, dilute 10 times to 10⁻¹⁰, and vortex to mix; (4) Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min; (5) Take 5 μL from each dilution centrifuge tube and add it to 2×YT solid medium (Amp), and incubate overnight at 37°C upside down; (6) The number of single colonies at different dilutions can be clearly distinguished on the statistical plate, and the number of phage particles per milliliter of phage solution, i.e., the phage library titer, is calculated according to the following formula:

[0049] Where T is the phage titer (unit: pfu / ml), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0050] 1.5.3 Amplification of the first round of phage elution buffer (1) The TG1 strain stored at -80℃ was streaked onto 2×YT solid medium and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium and incubated overnight at 37℃. (2) 500 μL of the overnight culture was transferred to 5 ml of 2×YT liquid medium and incubated at 37℃ and 220 rpm for about 45-60 min until the OD600 value was 0.5-0.55. (3) 500 μL of the phage elution buffer obtained after the first round of screening was added to the culture medium with an OD600 of 0.5-0.55. (4) The culture was incubated at 37℃ and 220 rpm for another 30 min. (5) The entire culture was evenly spread onto a medium containing 100 μg / ml of 2×YT liquid medium. (6) Incubate the overnight cultured square plate of Amp and 2% glucose in 2% agarose at 37°C overnight; (7) Take the overnight cultured square plate, add 6 ml of 2×YT liquid medium to the surface of the plate, gently scrape off the colonies with a spreader and collect the bacterial solution into a 15 ml centrifuge tube, which is the amplified bacterial sub-library. At the same time, measure the OD600 value of the bacterial solution with a spectrophotometer, add glycerol to a final concentration of 20%, which is the first round of bacterial library; (8) Calculate the corresponding bacterial solution volume of the eluted bacterial library according to the following formula, and transfer it to 100 ml of 2×YT liquid medium (containing 100 μg / ml Amp) so that the initial OD600 is 0.1:

[0051] Where V is the volume of the transferred bacterial solution (in μL), and OD600 is the OD600 of the constructed elution bacterial library; (8) Incubate at 37℃ and 220rpm until the OD600 of the bacterial solution reaches 0.5-0.55; (9) Calculate and add helper phage M13K07 according to the following formula to make the bacterial number: phage number = 1:20:

[0052] Where V is the volume of helper phage added (in ml), and T helper-phage (10) Continue culturing at 37℃ and 220rpm for 30 min; (11) Add Kana to a final concentration of 50μg / ml and incubate overnight at 30℃ and 220rpm.

[0053] 1.5.4 First round of phage purification (1) Transfer the overnight culture to a new 50ml centrifuge tube and centrifuge at 4000 rpm and 4℃ for 10 min; (2) Transfer the supernatant after centrifugation to a new 50ml centrifuge tube, add 1 / 4 volume of 20% PEG / 2.5M NaCl pre-cooled at 4℃, mix thoroughly and place on ice for 30min; (3) Centrifuge at 4000 rpm and 4℃ for 20 min, discard the supernatant, and invert on paper for 2 min; (4) Add 1 ml PBS to resuspend the precipitate, transfer the resuspended solution to a new 1.5 ml centrifuge tube, and centrifuge at 13000 rpm and 4℃ for 20 min. (5) Transfer the supernatant after centrifugation to a new 1.5ml centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix well and place on ice for 10min; (6) Centrifuge at 13000 rpm and 4℃ for 10 min, discard the supernatant, and resuspend the precipitate in 1 ml PBS; (7) Centrifuge at 13000 rpm and 4℃ for 2 min, and transfer the supernatant to a new 1.5 ml centrifuge tube, which is the first round of screening phage sub-libraries.

[0054] (8) First round of screening of phage sub-library titer detection.

[0055] 1.5.5 Multi-round screening The screening method is the same as the first round. The input phage is the phage sub-library obtained from the first round of screening, which is used as the input phage library for the second round of screening to obtain the phage elution buffer for the second round of screening. The titer of the second round phage elution buffer is detected, amplified, and purified in sequence. After detecting the titer of the screening phage sub-library, the third round of screening is performed.

[0056] 1.5.6 Monoclonal ELISA Detection (1) The TG1 strain stored at -80℃ was streaked on 2×YT solid medium and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5ml of 2×YT medium and incubated overnight at 37℃. (2) 500μL of the overnight culture was transferred to 5ml of 2×YT liquid medium and incubated at 37℃ and 220rpm for about 45min-60min until the OD600 value was 0.5-0.55. (3) 10μL of the phage elution buffer after the second round of screening was taken and serially diluted 10-fold in 1.5ml centrifuge tubes, for a total of 12 dilutions, and vortexed to mix. (4) 90μL of the bacterial solution with an OD600 value of 0.5-0.55 was added to each dilution centrifuge tube and mixed well. (5) Incubated at 37℃ and 220rpm for 30min. (6) The bacterial solution was evenly spread on a medium containing 100μg / ml of 2×YT liquid medium. (7) Randomly pick single colonies from the overnight culture plates and place them into sterile 96-well cell culture plates (P1-P2). Add 100 μL of 2×YT medium (containing 100 µg / ml Amp) to each well and incubate at 37°C overnight. (8) Transfer 2 μL of the overnight culture to a new 96-well cell culture plate with 220 μL of 2×YT liquid medium (containing 100 µg / ml Amp) per well and incubate at 37°C for 3 hours. (9) Calculate and add helper phage M13K07 to each well according to the following formula so that the bacterial count: phage count = 1:20:

[0057] Where V is the volume of helper phage added (unit ml), and Thelper-phage is the titer of the helper phage used; (10) Incubate at 37℃ for 30 min, add Kana to a final concentration of 50µg / ml, and incubate overnight at 30℃; (11) Centrifuge the 96-well plate after overnight incubation at 4℃, 4000rpm for 10 min, and store at 4℃ for later use; (12) Coat the enzyme-labeled plate with screening antigen (1ng / μL, coating solution is pH9.4 CBS, 100μL / well), and simultaneously coat with BSA as a control, and coat overnight at 4°C; (13) Discard the liquid in the overnight coated enzyme-labeled plate, add 200μL PBS buffer to each well, wash the enzyme-labeled plate 3 times at room temperature, 10 min each time; (14) Add 200μL blocking solution (3% BSA) to each well to block the enzyme-labeled plate, and block at room temperature for 1 h; (15) Discard the blocking solution, add 200μL of PBS buffer to each well. PBST (1×PBS plus 0.1% Tween 20, the same below) buffer, wash the microplate 3 times at room temperature, 10 min each time; (16) add 120 μL of 3% BSA to each well and then add 80 μL of the supernatant after centrifugation, incubate at room temperature for 2 h; (17) discard the liquid in the microplate, add 200 μL of PBST buffer to each well and wash 3 times, 10 min each time; (18) add M13 Bacteriophage Antibody (HRP), Mouse Mab, 1:8000 diluted in blocking buffer to each well, 100 μL / well, incubate at room temperature for 1 h; (19) discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well and wash 3 times, 10 min each time; (20) add 100 μL of TMB single-component colorimetric solution to each well, develop color in the dark for 2-3 min, add 100 μL of 1M The assay was terminated with HCl, and the OD450 value was read using a microplate reader, recorded, and saved. The results of the first monoclonal ELISA test are shown in Table 2.

[0058] Table 2 Results of the first monoclonal ELISA test

[0059] 1.5.7 Secondary ELISA validation of positive clones To rule out false positives, clones initially identified as positive underwent a second ELISA test, using the same method as above. The results of the second ELISA test for monoclonal clones are shown in Table 3.

[0060] Table 3 Results of the second ELISA assay for monoclonal antibodies

[0061] 1.5.8 Sequencing of positive clones Positive monoclonal antibodies were selected based on ELISA test data and secondary validation data.

[0062] Take 5 μL of positive clone bacterial culture from the monoclonal ELISA detection plate and inoculate it into 1 ml of 2×YT medium (containing 100 µg / ml Amp). Incubate at 37°C and 220 rpm until OD600 reaches 0.8-1.0 (approximately 6-8 h). Take 0.5 ml of bacterial culture for sequencing, and store the remaining bacterial culture at 4°C.

[0063] 1.5.9 Sequence Analysis Clones that were both positive in two single-clone ELISA verifications were ultimately classified as positive clones. According to this standard, a total of 192 positive clones were counted. These positive clones were sequenced, and 8 clones, Y1-6E-9A-9G-10B-10D and Y2-3C-3G-6C, failed to be sequenced. The remaining 184 sequences were translated into antibody sequences using software. Through sequence alignment analysis, a total of 49 different antibody sequences were obtained, and their nucleotide sequences are shown in SEQ ID NO.50-98. Sequencing results showed that clone Y1-1A was associated with clones Y1-1B, Y1-1E, Y1-1F, Y1-1G, Y1-2F, Y1-2G, Y1-3B, Y1-3C, Y1-3D, Y1-4B, Y1-4C, Y1-4D, Y1-4E, Y1-4F, Y1-5A, Y1-5C, Y1-5E, Y1-5G, Y1-5H, Y1-6G, Y1-7A, Y1-7B, Y1-7F, Y1-7H, Y1-8A, Y1-8B, Y1-8C, Y1-8D, Y1-8E, Y1-8F, Y1-9B, and Y1 -9C, Y1-9F, Y1-9H, Y1-10A, Y1-10C, Y1-10E, Y1-11B, Y1-11D, Y1-11G, Y1-12B, Y1-12C, Y1-12D, Y2-1B, Y2-1C, Y2-1D, Y2-1E , Y2-1H, Y2-2E, Y2-2F, Y2-2G, Y2-2H, Y2-3A, Y2-3B, Y2-3D, Y2-3E, Y2-3H, Y2-4B, Y2-4D, Y2-4E, Y2-4F, Y2-4G, Y2-4H, Y2-5A, Y2-5B, Y2-5C, Y2-5E, Y2-5F, Y2-5G, Y2-5H, Y2-6B, Y2-6H, Y2-7B, Y2-7C, Y2-7D, Y2-7F, Y2-7G, Y2-7H, Y2-8A, Y2-8B, Y2-8C, Y2-8D, Y2-8E, Y2-8F, Y2-8G, Y2-8H, Y2-9C, Y2-9D, Y2-9E, Y2-9G, Y2-9H, Y2-10B, Y2-10C, Y2-10D, Y2-10E, Y2-10F, Y2-11B, Y Clones Y2-11C, Y2-11D, Y2-11E, Y2-11F, Y2-11G, Y2-12A, Y2-12B, Y2-12C, Y2-12E, Y2-12G, and Y2-12H have the same nucleotide sequence; clone Y1-1H has the same nucleotide sequence as clones Y1-5D, Y1-6F, Y1-6H, Y1-11H, and Y2-6A; clone Y1-2A has the same nucleotide sequence as clones Y1-2D, Y1-2H, Y1-3A, Y1-4H, Y1-11F, Y2-4A, Y2-5D, and Y2-7E.Clones Y1-2B, Y1-6B, Y2-2A, and Y2-6D have the same nucleotide sequence; clone Y1-3E has the same nucleotide sequence as clones Y1-11C, Y2-1A, and Y2-10H; clone Y1-3F has the same nucleotide sequence as clone Y1-5F; clone Y1-5B has the same nucleotide sequence as clones Y2-6F and Y2-9F; clone Y1-7E has the same nucleotide sequence as clone Y2-2B; clone Y1-10G has the same nucleotide sequence as clones Y1-12H and Y2-10G; clone Y1-12E has the same nucleotide sequence as clone Y2-1F; and clone Y2-2D has the same nucleotide sequence as clone Y2-7A.

[0064] 2. Fermentation purification of nanobody sequences (1) Sequences were divided into antibody CDR regions using the Chothia rule and then aligned using MEGA11. (2) The sequence was synthesized by Beijing Anshengda Company and inserted between the Xbal and Xhol sites on the pET-32a plasmid to construct a recombinant plasmid. (3) The recombinant plasmid was transformed into TransB cells (purchased from Beijing TransGen Biotech Co., Ltd.) for expression and purification. (4) Single clones were selected for sequencing, and the correctly sequenced strains of glycerol bacteria were preserved. (5) Inoculate the bacterial culture into 5 ml of LB medium and incubate overnight at 37°C; transfer 1:100 to 5 mL LB tubes containing 1:1000 Amp and incubate overnight at 37°C; transfer 1:100 to two 200 ml LB medium bottles containing 1:1000 Amp and incubate at 37°C and 220 rpm until OD600 = 0.6~0.8; add IPTG to a final concentration of 1 mM and induce for 16 h at 16°C and 160 rpm. (6) Collect the bacterial cells by centrifugation at 4°C and 8,000 rpm for 30 min, and wash the precipitate 2~3 times with PBS solution. (7) Resuspend the collected bacterial cells in 1:20 PBS solution and sonicate them; the sonication conditions are: sonicate for 2 s, stop for 3 s, and sonicate for a total of 25 min. (8) Centrifuge at 4℃ and 12,000 rpm for 20 min to collect the supernatant of the broken solution. Use a syringe to draw the supernatant and filter it through a 0.45 μm PES membrane for later use. The single-point mutant nanobody was purified using the AKTA pure protein purification instrument. The specific steps are as follows: (1) Turn on the instrument and select the System Wash program. Rinse the ethanol in the instrument with deionized water. (2) After the program is completed, install a 5 ml nickel column on the instrument and rinse the ethanol in the column with 30 ml of deionized water until baseline equilibrium is reached. Set the flow rate to 5 ml / min and the upper limit of pressure to 0.5 MPa. (3) Replace the equilibration buffer and rinse the equilibration column. Set the flow rate to 3 ml / min. (4) After baseline equilibrium is reached, start loading the sample at a flow rate of 0.8~1.0 ml / min. After loading the sample, rinse slowly with 10 ml of equilibration buffer (flow rate is the same as loading the sample) and then rinse with a flow rate of 3 ml / min until baseline equilibrium is reached. (5) Elute with elution buffer, collect the elution peak protein, and set the flow rate to 3 ml / min; after all proteins are completely eluted, rinse the nickel column with deionized water until the baseline ion concentration is zero, and set the flow rate to 5 ml / min. (6) Continue rinsing with 50 ml of 20% ethanol solution to completely immerse the nickel column in ethanol, and then turn off the machine. Perform SDS-PAGE electrophoresis on the elution peak protein. Perform size exclusion experiment to further purify the protein, and the steps are as follows: (1) Use an ultrafiltration tube to concentrate the fraction obtained in the previous step, concentrate to 750 μL at 4 ℃ and 4000 rpm, and filter the concentrated solution through a 0.22 μm PES filter membrane.(2) Clean the internal tubing of the AKTA pure protein purifier using the system wash method. (3) After cleaning, install the Superdex™ 75 increase 10 / 300 GL pre-packed column onto the instrument and install a 500 μL injection loop on the instrument. Set the flow rate to 0.4 mL / min and the pressure limit to 2.8 MPa. (4) Using Load mode, wash the column with 1-2 column volumes of deionized water to level the baseline. Then, switch to PBS solution and equilibrate the column with 1-2 column volumes. At the same time, use a syringe to draw 5 mL of PBS solution and clean the injection loop through the injection hole. (5) Use a 1 mL syringe to draw the sample and inject it into the injection loop through the injection hole. Change the Load mode to Inject mode. (6) When the detector shows a UV absorption peak, start collecting until all peaks are collected. Change the Inject mode back to Load mode, continue rinsing the column and tubing, and then rinse with deionized water to level the baseline. (7) Use 20 Rinse the system with % ethanol, remove the column after 30 mL and store at 4℃, then turn off the instrument; (8) Perform SDS-PAGE gel electrophoresis experiment to analyze the purified and collected components.

[0065] 3. Surface plasmon resonance verification of the antigen-binding activity of nanobodies SPR assays are commonly used to accurately determine the affinity constant KD of antigen-antibody reactions. The instrument used in this experiment is the Cytiva BIAcore T200, chip model CM-5. The experiment consists of two steps: Amino-coupled antigen capture: (1) Install the CM5 chip on the instrument. Dilute the antigen to 50 μg / mL using the instrument's built-in sodium acetate solutions at pH 4.0, 4.5, and 5.0. Use the manual run program with a flow rate of 10 μL / min and an injection time of 120 s to pre-enrich the antigen and select the most suitable sodium acetate solution. (2) Dilute CXCL1 to 5 μg / mL using sodium acetate solution at pH 4.5. Use the Wizard program with the target level set to 2500 RU. Place the antigen diluent, 50 mM NaOH regeneration solution, carbodiimide / N-hydroxysuccinimide solution, and ethanolamine solution according to the program, and run the program. (3) When the RU value reaches the target level, the antigen is successfully coupled to the CM5 chip.

[0066] The Kinect and affinity procedure is used to determine the antibody KD value: (1) Use PBS solution to serially dilute the antibody to 200-0.78125 nM, and set the program for sample loading. The chip regeneration solution is 10 mM glycine solution (pH 2.0). (2) After the experiment, disconnect the instrument and import the obtained data into the instrument's software for analysis. Calculate the affinity constant K after data fitting. D Value. The SPR determination results for the affinity constant of nanobodies are shown in [reference needed]. Figure 4A , Figure 4B , Figure 4C , Figure 4D And Table 4.

[0067] Table 4 SPR Results

[0068] 4. Verification of the thermal stability of nanobodies by real-time quantitative PCR By setting a temperature gradient and detecting the exposure level of protein hydroxyl groups, the changes in protein spatial structure with temperature were analyzed to assess the thermal stability of the protein. The thermal stability of the nanobodies was evaluated using real-time quantitative PCR (qPCR) on a Thermo Fisher Scientific Q5 Real-Time PCR System. Three replicates were performed for each sample. After PCR, the data were imported into the instrument's software for analysis and calculation of the nanobodies' Tm values. Some thermal stability results are shown in Table 5.

[0069] Table 5 Thermal stability results

[0070] 5. Verify the blocking activity of nanobodies against CXCL1 HCT116 human colon cancer cells were divided into nine groups: 0 nmol / L CXCL1, 10 nmol / L CXCL1, 10 nmol / L CXCL1+Y1-3F, 10 nmol / L CXCL1+Y1-2C, 10 nmol / L CXCL1+Y1-8G, 10 nmol / L CXCL1+Y1-3E, 10 nmol / L CXCL1+Y1-1A, 10 nmol / L CXCL1+Y2-3F, and 10 nmol / L CXCL1+Corydalmine. Cells were digested according to cell passage procedures and resuspended. Cells were counted and seeded into appropriate culture dishes according to different experimental requirements. After plating, the cells were incubated at 37 ℃ in a 5% CO2 incubator. Once the cells adhered, the old culture medium was discarded, and the cells were gently washed with PBS. Different concentrations of exogenous CXCL1 (0 or 10 nmol / L) in 5% FBS medium were then added to the corresponding wells to treat the cells. For the antibody and blocking agent treatment groups, the antibody and blocking agent were added first, followed by the exogenous CXCL1. The cells were then cultured in the incubator.

[0071] like Figure 5 As shown, compared with the control group (0 nmol / L CXCL1), the colony formation rate of the 10 nmol / L CXCL1 control group was significantly increased, indicating that CXCL1 promotes colony formation. Furthermore, the colony formation rate of the positive control group (10 nmol / L CXCL1 + Corydalmine) was significantly reduced, indicating that blocking the CXCL1 / CXCR2 axis effectively inhibits the proliferation of HCT116 cells. In addition, compared with the control and 10 nmol / L CXCL1 control groups, the number of colonies in the antibody-treated groups was reduced, and this reduction showed a linear relationship with antibody affinity, indicating that the selected CXCL1 antibodies all possessed good biological activity.

[0072] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A nanobody against CXCL1, characterized in that, The nanobody contains a heavy chain variable region that targets CXCL1, and is composed of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; The amino acid sequence of the nanobody is shown in SEQ ID NO. 1, 2, 7, 11, 19, 33, 34, 37, 41, 43, 44, 45 or 48.

2. An isolated nucleic acid molecule, characterized in that, For a nucleic acid molecule or its complementary sequence that encodes the amino acid sequence of claim 1.

3. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The expression vector comprises the recombinant expression vector of claim 3, or the nucleic acid molecule of claim 2 integrated into the host cell genome; or the host cell expresses the anti-CXCL1 nanobody of claim 1.

5. An immunoconjugate, characterized in that, It includes the anti-CXCL1 nanobody as described in claim 1, and a conjugation portion, wherein the conjugation portion is a detectable marker.

6. A detection kit comprising the anti-CXCL1 nanobody of claim 1 and / or the immunoconjugate of claim 5; the kit may be used to detect the presence or level of CXCL1 in a sample.

7. A pharmaceutical composition, characterized in that, It includes the anti-CXCL1 nanobody as described in claim 1 or the immunoconjugate as described in claim 5.

8. Application of the nanobody of claim 1, the immunoconjugate of claim 5, the detection kit of claim 6, or the pharmaceutical composition of claim 7: preparation of a product for the treatment of colon cancer.

Images