Anti-CXCL1 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an anti-CXCL1 nano antibody and application thereof. The nano antibody comprises a heavy chain variable region targeting CXCL1, and the heavy chain variable region is composed of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; the amino acid sequence of the nano antibody and any one of SEQ ID NO.1-49 have at least 70% of sequence identity. The nano antibody has the advantages of small molecular weight, small immunogenicity and better solubility and stability, is beneficial to basic research and clinical tests, has potential diagnosis and treatment prospects for tumors, autoimmune diseases and neurological diseases, and has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nano-antibody against CXCL1 and application thereof. BACKGROUND

[0002] CXCL1 (C-X-C motif chemokine ligand 1), also known as GRO-alpha, is a chemokine (C-X-C subfamily) that mainly binds to the CXCR2 receptor. CXCL1 has important functions in physiology, including inducing angiogenesis and neutrophil recruitment, participating in inflammatory response and immune regulation, and is a key drug target for treating various tumors (such as pancreatic cancer, breast cancer, lung cancer, prostate cancer, liver cancer and colon cancer, etc.), autoimmune diseases (such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus and multiple sclerosis, etc.) and neurological diseases. Therefore, it is of great significance to design antibodies targeting CXCL1 for evaluating human health status, assessing the progression of related diseases, and carrying out targeted disease treatment.

[0003] However, the current detection and research of CXCL1 are mainly for inhibitors against CXCL1 or monoclonal and polyclonal antibodies against CXCL1. Reparixin is a non-competitive allosteric inhibitor of chemokine receptors CXCR1 and CXCR2; Corydalmine can alleviate the neuropathic pain caused by Vincristine by inhibiting the NF-κB-dependent CXCL1 / CXCR2 signaling pathway; NTC-001 is a humanized mouse anti-human CXCL1 antibody, and NTC-001 significantly inhibits tube formation in human umbilical vein endothelial cells (HUVEC). The existing small molecule inhibitors have defects such as poor targeting and toxic side effects, and full-length antibodies have effects but slow onset and are not easy to infiltrate deep tissues. Application in existing detection and related disease research and treatment still cannot meet the requirements, and the specificity and affinity still need to be improved. Therefore, it is particularly necessary to prepare a new type of antibody with good specificity and affinity. SUMMARY

[0004] In view of the above deficiencies of the prior art, the present application provides a nano-antibody against CXCL1 and application thereof. The present application successfully prepares a nano-antibody against CXCL1 with high affinity, stability and specificity, which can be used for detection and analysis of CXCL1 and diagnosis and treatment of diseases related to CXCL1 protein, especially diseases related to abnormally high expression of CXCL1. Based on the above research results, the present application is completed.

[0005] In a first aspect, the present application provides a Nanobody against CXCL1, which comprises a heavy chain variable region targeting CXCL1, consisting of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Preferably, the CDR region of the Nanobody can be composed of three CDR regions combined from any one of SEQ ID NO. 1-49 shown in Table 1.

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

[0007] Preferably, the amino acid sequence of the Nanobody has at least 70% sequence identity to any one of SEQ ID NO. 1-49; more preferably, at least 80% sequence identity to any one of SEQ ID NO. 1-49; further preferably, at least 90% sequence identity to any one of SEQ ID NO. 1-49; further preferably, at least 95% sequence identity to 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 application provides an isolated nucleic acid molecule capable of encoding the above-mentioned Nanobody against CXCL1.

[0009] The nucleic acid molecule sequence encoding antibody Y1-1A is: 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 the antibody Y1-1H is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCTGCTATTAGCTGGAGTGGTGGTACCACACACTATGCAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCCTCCCCCCGCACAAGGCATACTATGCCGGTACTTACTACTCTCCTTCAGAGTATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the 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 the antibody Y1-3F is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACTTCCCTTCAGTAGCTACGCCATGGGCTGGTTCCGCCAGGCTCTAGGGAAGGAGCGTGAGTTTGTCGCAGCGATTACTTGGAGTGGTGGTAGGACAGAGTTCGCAGACTCCGTGCAGGGCCGAGGCACCATCTCCAGAGACAACGACAAGAGCACGGTGTCTCTAACAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCAGCGGATCAGAGTCCCGTCCCCTACTACGAGGGAGAGGGCTGGGAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the 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 the antibody Y1-4G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGCATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCCGCCTCTGGACGCCCCATCAATAACTATGCCATGACCTGGTTCCGCCGGGCCCCAGGGAAGGAGCGTGAGTTTGTAGCAATAATTAATAACAGTGGTACGATCACTTACTATACAGAGCGCGTGAAGGGTCGATTCACCATCTCCAGAGACAACGACCTGAGCACGGTGTTTCTGCAAATGAACGACCTGAAACCGGAGGACACGGCCGTTTATTACTGCGCAGCCGGAGTGCGGAATTATCGTCCCCTTAGTACCTACACCCCGCGTGACTTCGTTTTCTGGGGTCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-5B is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGACTCTCTGACACTCTCCTGCGCGGACTCTGGACGCGCCACGGATTCATATTCCGTGGGCTGGTTCCGCCAGGCTCCAGGAAAGGACCGTGAGTTTGTGGCAGTCATTAGCTGGATTGCAGGTATCACATATTATGCAGAATCCATTCAGGGCCGATTCGTCGTCTCCAGAGACAACGCCAAGAACACGGTGTCTCTACAAATGAACAGCCTAAAACCCGAGGACACGGCCGTTTATTACTGTGCAGCCCAACCTAGGAAAACCTGGTACCGGGCGGCGGAAGAGGATCGCTATTCGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-6A is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGGTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTTCGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAACTGGAGTGGTACTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCGCCAAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTAGCGGATTGGGGTACGTAACCCCCGGCGAATATGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-6C is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGACCGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGATGATTATGCCATCGGCTGGTTCCGCCAGGCCCCGGGGAGGGGGCGTCAGGGGGTCGCATGCATTAGTGCTGCTGATGGTAGTATATACTATGGACACTCCGTGAAGGGCCGATTCACCATGTCCAGTGACAGCGCCAACAACATGTTCTATCTAGAAATGAATATGCTACAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGATCTAATTTGCCCTTTGGGATTGGGACACGAATTGAGATATTCGCATGACTACTCGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-6D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCCGCCTCTGGACGCACCATCAGTAGTACTGCCATGGCATGGTTCCGCCAGGCTCCAGGGAAGGGACGTGAATTTGTAGCCGCTATTAGGTGGAGTGATGGAGACACATCTTATACAAGTAACGTGCAGGACCGATTCATTATCTCCAGAGACAACGCCAACAGCACCATGTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGCACGCCCAACCGCTAGTACTAGGCTCGTCTACATTAGGGACTATGAGTATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-7C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCCATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTCGGTCGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTAGTTGGAGTGGTGGTAACACGGAGTATGCAGACTCCGTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACACGGTGTATCTACGTATGAACAACCTGATCCCTGAGGACTCGGCCGTTATTACTGTGCAGCAGATTCATCCCCGACGCCCTTTTGGGTAGTAGGTAGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Y1-7E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCATCTTCAGTAGCGCTGCCATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTGGTAGCACGTATACTCTGGAGCGGTGGTAGCACATACTATGAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTATGTGCAGTAGCAAAGTCCTACGCGCCGTTTCGCGATGTTTCTTCTTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-7G is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCCCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAACTGGAGTGATGGTAAGTCATTCTATGCAGACTCCGTGAAGGGCCGAGTCACCGTCTCCAGAGACAACGCCAAGAACACGGGGTATCTGCAAATGAACAGCTTGAAACCTGAGGATACGGCCGTTTATTACTGTGCAGCTGATCGGGAATTATACTATAGTGGTAGTTACTACCGCGCCGAGGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the 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 the antibody Y1-9E is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTACCGATACCATGGCCTGGTACCGCCAGGCCCCAGGGAAGCAGCGCGCATTGGTCGCAACTATTGCAAGTGATAGTAGAACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGCCGTCTATTACTGCAAAGGCATAATTAGAGATAGCTGGTACGCCCCTCTCGCGGACTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y1-10F is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGCGTCTCCTGTGCAGCCTCTGGTCTTACCTTCAGTAACTACACCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCAGTTATTGGTAGGAGTGGTCGTTACTCAAGGTATGCAGACTCCGTGAAGGGCCGATTCATCATCTCCAGAGATAACACCAAGAGCAACGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACTGGCGTTTATTACTGTGCAGCCGCCCTGATATTCAGGTTAGATCCAAAGCCTGACTATTGGGGCCAGGGAACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-10G is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCATTTTCAGTACCTATGGCGTGGGCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTGCTAGGAATGGTGGAAACATACAGTATGGGGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTTAGTCCAACTCCCTACTATAGGGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-10H is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATATAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-11A is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGTCATCTCTGAGACTCTCCTGCGCAGCCTCTTCTGTAGCCTCTGAACGCACCCTCAACGACTACACCGTGGGCTGGTTCCGCCAGGGTCCAGGGACGGAGCGTGAATTTGTAGCAGCGGTTAGTTGGCTCCGTGAGAATACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAACCTGAAACGTGAGGACACGGCCGTTTATTATTGCGCAACCCGGACGGGAAGTCTTACTTCGCGGACGGAATATGAGTACGACTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-11E is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGAGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAACTATTAATAGTGGTGGTGGTATCACAACCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTGTATTACTGTACAAAAGCGGGGGCGAGCGACCCCCCCCCGGGGCCAGGGGACCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-12A is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCTTAGGTCGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTGCGTGAGTTTGTAGGAGGCATTAGTTGGAGTGGTGGTAACACGGAGTATGCAGACTCCGTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACACGGTGTATCTACGTATGAACAACCTGATCCCTGAGGACTCGGCCGTTTATTACTGTGCAGCAGATTCATCCCCGACGCCCTTTTGGGTAGTAGGTAGTTGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding antibody Yl-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 the antibody Y2-1G is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGATGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTACACGCACCTTTAGTAGATATGTTATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGACTTTGTAGCAGCTATTAGCAGGACTGCTAATGCAGTCCATACAGACTCCGTGAAGGGTCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGGTGTCGGGGGATACTATGGTAGTATTGAGGGGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y2-2C is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGGGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG; The nucleic acid molecule sequence encoding the antibody Y2-2D is: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGTAGCATCAGTCGTCAAGCCATGGGCTGGTTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAACGGCTATGAACTACAATGGTGAAAGTATGGTCTATGCAGCATTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATACGGTGTATCTACAAATGGACAGCCTGAAACCTGAAGACACGGCCGTATATTATTGTGCGGCAGGTCACTACGGATTGAGGTACGACATGTCGGCCGTGAGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG The nucleic acid molecule sequence encoding the 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 the antibody Y2-12D is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAATCTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTATCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAAAGCGTGACTTTATAGCAGCGATTAGTAGGGGTGGTGGTAGCACATTCTATGGAGACTCCGTGCTGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGCCTATCTGCACATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATATTAGTCCAACTCCCTACTATAAAGATTATGCGTGGGCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG The nucleic acid molecule sequence encoding the antibody Y2-12F is: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCCGGATTCACCTTCGGTAATTATTGGATGCATTGGGTCCGTCAGGCTCCGGGGAAGGGGCTCGAGTGGATCTCGGGAATAAATGAGGCGAATAGTGAAACATGGTATGGAGACGCCGTACAGGGCCGATTCACCATCTCCAGAGACAACTCCAGGAACACGCTACATCTGCAATTAATGAGTCTGAAGGCTGACGATTCGGCCAAGTATTACTGTGTGAGAGACCGCGGGAATTACGGTGGATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGG.

[0010] In a third aspect, the present application provides a recombinant expression vector, which comprises the nucleic acid molecule as described above.

[0011] The recombinant expression vector is obtained by linking the nucleic acid molecule with an expression vector; wherein the expression vector can be selected from DNA, RNA, viral vector, plasmid, bacteriophage, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, transposon, other gene transfer system, or a combination thereof. In a fourth aspect of the present application, a host cell comprising the above-mentioned recombinant expression vector, or comprising the above-mentioned nucleic acid molecule integrated into the genome of the host cell, is provided; or the host cell expresses the above-mentioned anti-CXCL1 nanobody.

[0012] Further, the host cell includes cells of prokaryotic and eukaryotic origin. The prokaryotic is preferably Escherichia coli, and the eukaryotic includes yeast and mammalian cells, wherein the mammalian cells include CHO, NS0, HEK293, PERC6, etc., which are not specifically limited herein.

[0013] In a fifth aspect of the present application, an immunoconjugate is provided, which comprises the anti-CXCL1 nanobody of the present application, and a conjugated moiety, wherein the conjugated moiety is a detectable label, a drug, a toxin, a cytokine, a viral coat protein or a virus-like particle, etc.

[0014] The detectable label includes a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0015] The drug can be a cytotoxic drug, such as an anti-tubulin drug, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, etc.

[0016] The toxin can be aureus, maytansinoid, ricin, ethidium bromide, mitomycin, diphtheria toxin, abrin, gelonin, maytansinol, restrictocin, phenomycin, curcin, tetrodotoxin, calicheamicin, glucocorticoid, etc., which are not specifically limited herein.

[0017] The immunoconjugate contains a multivalent (such as bivalent) anti-CXCL1 nanobody as described herein. The multivalent refers to the inclusion of multiple repeats of the anti-CXCL1 nanobody as described herein in the amino acid sequence of the immunoconjugate.

[0018] In a sixth aspect of the present application, a conjugate is provided, which comprises the above-mentioned anti-CXCL1 nanobody, and a conjugated moiety; preferably, the conjugated moiety is selected from a protein tag, a detectable label, a therapeutic agent or another biologically active polypeptide.

[0019] The protein tag comprises a purification tag; the detectable label comprises 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 comprises any one of an anti-tumor drug, an anti-inflammatory drug, or an immunosuppressant.

[0020] In a seventh aspect, the present application provides a detection kit comprising the anti-CXCL1 nanobody, the immunoconjugate, or the conjugate described above; the kit can be used for detecting the presence or level of CXCL1 in a sample.

[0021] The kit can be used for non-invasively detecting the expression of CXCL2 in a subject.

[0022] In an eighth aspect, the present application provides a pharmaceutical composition comprising the composition, the immunoconjugate, or the conjugate described above.

[0023] Further, it can further comprise a pharmaceutically acceptable carrier and / or excipient. Moreover, according to the usual methods, it can be prepared into a dosage form of a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a spray, etc. for oral administration, a dosage form for external use, a suppository, and a sterile injection solution.

[0024] The carrier and / or excipient, etc. that can be contained are well known in the art, and a person of ordinary skill in the art can determine that they meet the clinical standards.

[0025] Further, the pharmaceutical composition of the present application can be administered into the body by known means. For example, it can be delivered into the body systemically through intravenous injection or locally into the tissue of interest. Alternatively, it can be administered intravenously, transdermally, intranasally, mucosally, or by other delivery methods. Such administration can be performed via a single dose or multiple doses. A person skilled in the art understands that the actual dose to be administered in the present application can vary greatly depending on various factors, such as the target cells, the biological type or tissue thereof, the general condition of the subject to be treated, the administration route, the administration mode, etc.

[0026] The subject to which the pharmaceutical composition is administered can be a human and a non-human mammal, such as a mouse, a rat, a guinea pig, a rabbit, a dog, a monkey, a chimpanzee, etc. Among them, a human is the most preferred.

[0027] In a ninth aspect, the present application provides the use of the anti-CXCL1 nanobody, the immunoconjugate, the conjugate, the kit, or the pharmaceutical composition described above in any one or more of the following: (a1) preparing a product for detecting, monitoring, diagnosing, and / or prognosing a tumor or an autoimmune disease or a neurological disease; (a2) preparing a product for preventing and / or treating a tumor or an autoimmune disease or a neurological disease; The tumor or the autoimmune disease or the neurological disease is characterized by abnormally high expression of CXCL1.

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

[0029] In a tenth aspect, the application provides use of the anti-CXCL1 nanobody in the preparation of a fusion protein.

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

[0031] The application also discloses a screening method of the anti-CXCL1 nanobody, which specifically comprises the following steps: (1) immunizing a llama with a recombinant CXCL1 protein; (2) isolating peripheral blood lymphocytes of the llama, extracting total RNA and reverse transcribing the total RNA to synthesize cDNA; (3) using primers to perform two rounds of PCR amplification on a target fragment, constructing a phagemid, transferring the phagemid into a bacteriophage to construct a bacteriophage library, and eliminating positive clones in multiple rounds, wherein the primers are as follows: First round of primers: CALL 001: GTCCTGGCTGCTCTTCTACAAGG; CALL 002: GGTACGTGCTGTTGAACTGTTCC; second round of primers: VHH-For: GACTAGTCACTAGTGATGTGCAGCTGCAGG; VHH-Back: CGAGCTCGGAGCTCTGAGCGG.

[0032] (4) constructing an expression vector for the positive clone, using a prokaryotic expression system to induce expression and purification, and determining the affinity, thermal stability and biological activity of the positive clone to CXCL1, so as to complete screening of the anti-CXCL1 nanobody.

[0033] The application provides an amino acid sequence of a nanobody that specifically and highly binds to CXCL1, as well as a construction method and an expression and purification strategy thereof. Those skilled in the art can refer to the content herein and appropriately improve process parameters.

[0034] Compared with the prior art, the technical scheme of the application has the following beneficial effects: The application adopts the recombinant CXCL1 to immunize the llama, establishes the nanobody phage library, and screens out the nanobody sequence capable of high affinity binding of CXCL1, has small molecular weight, small immunogenicity, better solubility and stability, is favorable to basic research and clinical test, has potential diagnosis and treatment prospects for tumors, autoimmune diseases and neurological diseases, and has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the total RNA agarose gel electrophoresis result of llama PBMC in the embodiment of the application; Figure 2 It is the random colony PCR agarose gel electrophoresis result of the bacterial library in the embodiment of the application; Figure 3A It is the SDS-PAGE electrophoresis result of the antibody expressed and purified by part of TransB in the embodiment of the application; Figure 3B It is the SDS-PAGE electrophoresis result of the antibody expressed and purified by part of TransB in the embodiment of the application; Figure 4A It is the schematic diagram of the result of SPR determination of the nanobody affinity constant in the embodiment of the application; Figure 4B It is the schematic diagram of the result of SPR determination of the nanobody affinity constant in the embodiment of the application; Figure 4C It is the schematic diagram of the result of SPR determination of the nanobody affinity constant in the embodiment of the application; Figure 4D It is the schematic diagram of the result of SPR determination of the nanobody affinity constant in the embodiment of the application; Figure 5 It is the influence of the nanobody on the clone formation of HCT116 cells in the embodiment of the application. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, 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 application belongs.

[0037] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, they indicate the presence of a feature, step, operation and / or combination thereof.

[0038] EMBODIMENT 1. Immunization, library construction and screening of anti-CXCL1 Nanobody The antigen used for immunizing the alpaca was recombinant CXCL1 protein. The antigen was injected around the neck lymph nodes of the alpaca, and the alpaca was immunized once every fourteen days. The peripheral blood of the immunized alpaca was extracted 5 mL each time, and the plasma was separated for antibody titer determination. A total of five immunizations were performed, and the plasma titer reached 10 4 .

[0039] 1.1 RNA extraction and reverse transcription (1) The peripheral blood lymphocytes stored in Trizol were transferred to a 1.5 mL centrifuge tube, and 1 / 5 volume of chloroform was added and mixed well; (2) After standing at room temperature for 5 minutes, centrifugation was performed at 4°C 12000g for 15 minutes; the supernatant after centrifugation was carefully transferred to a new centrifuge tube; an equal volume of isopropanol was added to the new centrifuge tube; after standing at room temperature for 10 minutes, centrifugation was performed at 4°C 12000g for 10 minutes; (3) The precipitate in each tube was washed with 1 mL of 75% ethanol, and after centrifugation at 7500g for 5 minutes, the ethanol was removed and dried. The precipitate was dissolved in an appropriate amount of RNase-free water, and all samples were combined to obtain the total RNA; (4) The total RNA obtained was reverse transcribed using a Takara reverse transcription kit. The total RNA sample was divided into two parts, one used Oligo dT Primer in the kit as primer, and the other used Random 6-mers in the kit as primer. The total RNA obtained in the previous step was reverse transcribed into cDNA according to the instructions of the reverse transcription kit, and was saved in two centrifuge tubes, respectively.

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

[0041] Second round PCR amplification: (1) The first round of PCR amplification product as a template for the second round of PCR reaction, the second round of amplification primer as follows: VHH-For: GACTAGTCACTAGTGATGTGCAGCTGCAGG; VHH-Back: CGAGCTCGGAGCTCTGAGCGG. In order to determine the optimal template usage, respectively using 0.3 μL, 0.5 μL, 1 μL, 2 μL, 3 μL as a template, according to the following configuration reaction system: the first round of PCR recovery product 0.3-3 μL, VHH-For / VHH-Back 2 μL / 2 μL, dNTP Mix 4 μL, 10×ExTaqBuffer 5 μL, HS Ex Taq 0.25 μL, ddH2O Up to 50 μL. Reaction conditions: step 1, 98℃, 3 min; step 2, 94℃, 50 s, 55℃, 30 s, 72℃, 40 s; go to step 2, 11 cycles; step 3, 72℃, 5 min; step 4, 4℃, forever. (2) After the reaction, take 20 μL PCR product for 1% agarose gel electrophoresis, finally select the electrophoresis results in the purpose of band single and fragment size of 400 bp or so of the template amount is the best template amount, the first round of PCR amplification product of about 1 / 5 volume of 288 reactions according to this template amount and using 1.2 (1) the same conditions for PCR reaction, then using universal type DNA purification recovery kit for DNA purification of PCR reaction liquid; (3) the recovery product is collected into a centrifugal tube, which is the second round of PCR amplification product, at the same time, 2 μL is taken to detect the concentration of the recovery product by nucleic acid concentration measuring instrument and recorded, and the rest of the product is stored at -20℃.

[0042] 1.3 Enzymatic digestion and ligation Vector and PCR product enzymatic digestion: (1) Using pComb3XSS as phage plasmid vector, using restriction endonuclease Spe I, Sac I respectively digest 12 μg pComb3XSS vector and 4 μg second round of PCR amplification product, 37℃ incubation for 4 h; (2) using DNA recovery purification kit to purify pComb3XSS vector and second round of PCR amplification product, 4℃ storage. Vector and fragment ligation, reaction system 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) The ligation reaction is incubated at 4℃ overnight (about 16 h); (4) using universal type DNA purification recovery kit to purify the ligation reaction liquid, detecting the concentration of the recovery product, 4℃ storage.

[0043] 1.4 Bacterial library and phage library construction 1.4.1 Verification of transformation efficiency of ligation product (1) Take a 50 μL of TG1 competent cells on ice for 5-10 min to thaw; (2) add 100 ng of ligation product, transfer to the pre-cooled 1 mm spacing electrotransformation cup, set the parameters in the electrotransformation instrument: 1800V, 1 mm, click the button to click transformation; (3) immediately add 1 ml of 37°C preheated SOC culture solution after waiting for the completion of the electrotransformation, mix well and recover at 37°C, 220 rpm for 1 h; (4) take 100 μL from the recovered bacterial solution for 10-fold gradient dilution and plate, calculate the number of transformed bacteria obtained from each reaction according to the dilution fold and the number of single bacteria, which is the transformation efficiency of the ligation product; (5) at the same time, randomly select 48 single colonies for colony PCR, and the PCR product with a single band of about 400 bp is considered as a positive clone, thereby estimating the single colony positive rate.

[0044] 1.4.2 Bacterial library construction (1) Take 24 100 ng ligation systems and use 24 tubes of competent cells for electrotransformation according to the above method; (2) after recovering at 37°C for 1 h, take 100 μL from them for 10-fold gradient dilution and plate, and incubate at 37°C overnight; (3) collect all the remaining bacterial solutions and evenly spread them on 5 245 mm square culture plates (2 x YT containing 100 μg / ml Amp, 2% glucose, 2% agarose) and incubate at 37°C overnight; (4) calculate the number of transformed bacteria obtained from all reactions according to the dilution fold and the number of single bacteria, which is the library capacity of the bacterial library; (5) at the same time, randomly select 48 single colonies from the gradient dilution plate for colony PCR to verify the cloning positive rate of the bacterial library; (6) scrape the colonies on the overnight incubated 245 mm square culture plate using 2 x YT liquid medium, place them in a 50 ml centrifuge tube, measure the OD600 value, and add 20% glycerol to a final concentration and store at -80°C.

[0045] 1.4.3 Preparation of phage library (1) According to the OD600 of the bacterial library, calculate the volume of the bacterial library that needs to be added to 100 ml of 2 x YT liquid medium, inoculate the bacterial library into 100 mL of 2 x YT liquid medium (containing 100 μg / ml Amp) according to the calculation result, incubate at 37°C, 250 rpm until the OD600 is 0.5-0.55; (2) according to the titer of the helper phage, add the helper phage at a ratio of 1:20 (number of bacteria: number of phages), incubate at 37°C, 250 rpm for 30-60 min; (3) add Kana to a final concentration of 50 μg / ml, incubate at 30°C, 250 rpm overnight. (4) centrifuge the overnight culture at 8000 rpm for 10 min at 4°C, then transfer the supernatant to a new 50 ml centrifuge tube; (5) add 1 / 4 of the pre-cooled PEG / NaCl stock solution, mix well, and incubate on ice for 30 min; (6) centrifuge at 8000 rpm for 10 min at 4°C, discard the supernatant, and invert to dry for 2 min; (7) resuspend in a new centrifuge tube with 5 mL PBS, centrifuge at 8000 rpm for 10 min at 4°C; (8) transfer the supernatant to a new centrifuge tube after centrifugation, and add 1 / 4 volume of pre-cooled PEG / NaCl stock solution again, mix well, and incubate on ice for 10 min; (9) centrifuge at 8000 rpm for 10 min at 4°C, discard the supernatant, resuspend with 1 mL PBS, and centrifuge at 8000 rpm for 10 min, then transfer the supernatant to a new centrifuge tube and store at -80°C, which is the purified phage library.

[0046] 1.5 Immunological screening 1.5.1 First round of screening (1) Take the screening antigen out of the -80°C freezer and thaw on ice; (2) coat the immunotubes with the screening antigen (50 μg / tube, coating solution is PBS, 2 ml / tube), and at the same time, coat the milk control, slowly rotate at 4°C overnight; (3) discard the liquid in the immunotube after overnight coating, add 2 ml PBS buffer, and wash the immunotube at room temperature for 3 times, each time rotating for 5 min; (4) add 2 ml blocking solution (3% skim milk powder) solution, and block at room temperature for 2 h; (5) discard the liquid in the immunotube after blocking, add 2 ml PBST buffer, and wash the immunotube at room temperature for 3 times, each time rotating for 5 min; (6) discard the washing solution in the immunotube, add 2 ml PBS, and calculate the prepared phage library according to the following formula and add it as the first round of screening input phage library, and incubate at room temperature for 1 h:

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

[0048] 1.5.2 Titer detection of the first round of phage eluate (1) The TG1 strain stored in a -80°C refrigerator was streaked on a 2xYT solid medium and incubated at 37°C overnight (stored at 4°C for one week), and one single colony was picked from the single colony plate to 5 ml of 2xYT medium and incubated at 37°C overnight; (2) 500 μL of the overnight culture was transferred to 5 ml of 2xYT liquid medium, and incubated at 37°C, 220 rpm for about 45-60 min until the OD600 value was 0.5-0.55; (3) 10 μL of the first round of phage eluate was diluted by 10 times in a 1.5 ml centrifuge tube, and a total of 10 gradients were diluted to 10-10, and shaken to mix; (4) 90 μL of TG1 bacterial liquid was added to each diluted centrifuge tube, mixed, and incubated at 37°C for 30 min; (5) 5 μL was taken from each diluted centrifuge tube and added dropwise to 2xYT solid medium (Amp), and incubated at 37°C overnight; (6) the number of single colonies that could be clearly distinguished in the dilution on the plate was counted, and the number of phagemids per milliliter of phage solution was calculated according to the following formula, that is, the titer of the phage library:

[0049] wherein T is the titer of the phage (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 eluate (1) The TG1 strain stored in a -80°C refrigerator was streaked on a 2xYT solid medium and incubated at 37°C overnight (stored at 4°C for one week), and one single colony was picked from the single colony plate to 5 ml of 2xYT medium and incubated at 37°C overnight; (2) 500 μL of the overnight culture was transferred to 5 ml of 2xYT liquid medium, and incubated at 37°C, 220 rpm for about 45-60 min until the OD600 value was 0.5-0.55; (3) 500 μL of the first round of phage eluate obtained after screening was added to the bacterial liquid with an OD600 value of 0.5-0.55; (4) 37°C, 220 rpm continued to culture for 30 min; (5) the whole bacterial liquid was evenly spread on a 245 mm square medium plate containing 100 μg / ml Amp and 2% glucose in 2% agarose, and incubated at 37°C overnight; (6) the square plate was taken after overnight culture, 6 ml of 2xYT liquid medium was added to the surface of the culture plate, and the colonies on the square plate were scraped off with a spreader and collected in a 15 ml centrifuge tube, which was the amplified bacterial sub-library. At the same time, the OD600 value of the bacterial liquid was measured using a spectrophotometer, and glycerol was added to a final concentration of 20%, which was the first round of bacterial library; (7) the eluate bacterial library was calculated according to the following formula to transfer to 100 ml of 2xYT liquid medium (containing 100 μg / ml Amp) to make the initial OD600 value 0.1:

[0051] Wherein, V is the volume of the adapter bacteria liquid (unit μL), OD600 is the OD600 of the constructed eluent bacteria library; (8) 37°C, 220 rpm culture until the OD600 of the bacteria liquid reaches 0.5-0.55; (9) calculate and add the helper phage M13K07 according to the following formula to make the number of bacteria: the number of phages = 1:20:

[0052] Wherein, V is the volume of the added helper phage (unit ml), T helper-phage is the titer of the used helper phage; (10) 37°C, 220 rpm continue to culture for 30 min; (11) add 50 μg / ml Kana with a final concentration, 30°C, 220 rpm overnight culture.

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

[0054] (8) Titer detection of the first round of phage sub-library screening.

[0055] 1.5.5 Multiple rounds of screening The screening method is the same as the first round, and the input phage is the phage sub-library obtained in the first round of screening, which is used as the input phage library in the second round of screening to obtain the second round of phage eluate. The second round of phage eluate titer detection, amplification and purification are sequentially performed, and the sub-library of the screened phage is detected for titer before the third round of screening.

[0056] 1.5.6 Monoclonal ELISA detection (1) The TG1 strain stored in a -80°C refrigerator was pre-streaked on a 2xYT solid culture medium and cultured overnight at 37°C (stored at 4°C for one week), and a single colony was picked from the single colony plate to 5 ml of 2xYT medium and cultured overnight at 37°C; (2) 500 μL of the overnight culture was transferred to 5 ml of 2xYT liquid medium, and cultured at 37°C, 220 rpm for about 45-60 min until the OD600 value was 0.5-0.55; (3) 10 μL of the phage eluate after the second round of screening was diluted 10-fold in a 1.5 ml centrifuge tube, a total of 12 gradients were diluted, and shaken to mix well; (4) 90 μL of the bacterial solution with an OD600 value of 0.5-0.55 was added to each dilution centrifuge tube and mixed well; (5) continue to culture at 37°C, 220 rpm for 30 min; (6) evenly spread the bacterial solution on a solid culture medium plate containing 100 μg / ml Amp and culture overnight at 37°C; (7) randomly pick single colonies from the overnight culture medium plate to a sterile 96-well cell culture plate (P1-P2), and add 100 μL of 2xYT medium (containing 100 μg / ml Amp) to each well and culture overnight at 37°C; (8) take 2 μL of the overnight culture and transfer it to a new 96-well cell culture plate containing 220 μL of 2xYT liquid medium (containing 100 μg / ml Amp) per well, and culture at 37°C for 3 h; (9) calculate and add helper phage M13K07 to each well according to the following formula to make the number of bacteria: phage number = 1:20:

[0057] Wherein, V is the volume of the helper phage added (unit: ml), Thelper-phage is the titer of the helper phage used; (10) incubate at 37℃ for 30 min, add Kana with a final concentration of 50 µg / ml, and incubate at 30℃ overnight; (11) centrifuge the 96-well plate after overnight incubation at 4℃ and 4000 rpm for 10 min, and store at 4℃ for standby; (12) coat the enzyme-labeled plate with the screening antigen (1 ng / μL, the coating solution is CBS with pH 9.4, 100 μL / well), and coat with BSA as a control at the same time, and coat at 4°C overnight; (13) discard the liquid in the enzyme-labeled plate after overnight coating, add 200 μL of PBS buffer to each well, and wash the enzyme-labeled plate at room temperature for 3 times, each for 10 min; (14) add 200 μL of blocking solution (3% BSA) to each well to block the enzyme-labeled plate at room temperature for 1 h; (15) discard the blocking solution, add 200 μL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer to each well, and wash the enzyme-labeled plate at room temperature for 3 times, each for 10 min; (16) add 120 μL of 3% BSA to each well, and then add 80 μL of the supernatant after centrifugation, and incubate at room temperature for 2 h; (17) discard the liquid in the enzyme-labeled plate, wash each well with 200 μL of PBST buffer for 3 times, each for 10 min; (18) add M13 Bacteriophage Antibody (HRP), Mouse Mab, diluted in blocking solution at 1:8000 to each well, 100 μL / well, and incubate at room temperature for 1 h; (19) discard the liquid in the ELISA plate, wash each well with 200 μL of PBST buffer for 3 times, each for 10 min; (20) add 100 μL of TMB single-component color developing solution to each well, develop color in the dark for 2-3 min, add 100 μL of 1M Hcl to each well to stop the reaction, and read the OD450 value by using an enzyme-labeled instrument, and record and save. The first ELISA detection results of the monoclonal are shown in Table 2.

[0058] Table 2 First ELISA detection results of the monoclonal

[0059] 1.5.7 Secondary verification of positive clones by ELISA In order to exclude false positive results, the clones preliminarily identified as positive were subjected to secondary verification by ELISA, and the detection method was the same as above. The second ELISA detection results of the monoclonal are shown in Table 3.

[0060] Table 3 Second ELISA detection results of the monoclonal

[0061] 1.5.8 Sequencing of positive clones The positive monoclonal was selected according to the ELISA detection data and the secondary verification data.

[0062] From the positive clone well of the monoclonal ELISA detection plate, 5 μL of bacteria liquid was inoculated into 1 ml of 2xYT medium (containing 100 μg / ml Amp), and cultured at 37°C, 220 rpm until OD600was 0.8-1.0 (about 6-8 h). 0.5 ml of bacteria liquid was taken for sequencing, and the rest of the bacteria liquid was stored at 4°C.

[0063] 1.5.9 Sequence analysis The clones determined as positive clones by twice monoclonal ELISA verification results were finally determined as positive clones. According to this standard, a total of 192 positive clones were counted, and the positive clones were sequenced. Among them, Y1-6E-9A-9G-10B-10D, Y2-3C-3G-6C, 8 clones failed to sequence, and 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 the nucleotide sequences are shown as SEQ ID NO. 50-98. The sequencing results show that clone Y1-1A has the same nucleotide sequence as clone 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, 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, Y2-11C, Y2-11D, Y2-11E, Y2-11F, Y2-11G, Y2-12A, Y2-12B, Y2-12C, Y2-12E, Y2-12G, Y2-12H; clone Y1-1H has the same nucleotide sequence as clone Y1-5D, Y1-6F, Y1-6H, Y1-11H, Y2-6A; clone Y1-2A has the same nucleotide sequence as clone Y1-2D, Y1-2H, Y1-3A, Y1-4H, Y1-11F, Y2-4A, Y2-5D, Y2-7E;Clone Y1-2B has the same nucleotide sequence as clones Y1-6B, Y2-2A, Y2-6D; clone Y1-3E has the same nucleotide sequence as clones Y1-11C, Y2-1A, 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, 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, Y2-10G; clone Y1-12E has the same nucleotide sequence as clone Y2-1F; clone Y2-2D has the same nucleotide sequence as clone Y2-7A.

[0064] 2 Fermentation purification of nanobody sequences (1) The sequenced sequence is separated by the chothia rule for antibody CDR region, and then the sequence alignment is performed by MEGA11. (2) The sequence is synthesized by Beijing Anshengda Company and inserted into the Xbal and Xhol sites of pET-32a plasmid to construct a recombinant plasmid. (3) The recombinant plasmid is transformed into the expression and purification of TransB cells (purchased from Beijing Quanshi Gold Company). (4) Single colonies are picked for sequencing, and glycerol bacteria of correct strains are saved. (5) Inoculate the bacterial liquid with 5 ml of LB medium, and incubate at 37°C overnight. Transfer 1:100 to 5 mL of LB medium containing 1:1000 Amp, and incubate at 37°C overnight. Transfer 1:100 to 2 bottles of 200 ml of LB medium containing 1:1000 Amp, and incubate at 37°C, 220 rpm until OD600=0.6~0.8. Add IPTG with a final concentration of 1 mM, and induce at 16°C, 160 rpm for 16 h. (6) Centrifuge at 4°C, 8,000 rpm for 30 min to collect the bacterial body, and wash the precipitate with PBS solution for 2~3 times. (7) Resuspend the centrifuged bacterial body with 1:20 PBS solution, and perform ultrasonic crushing. The crushing conditions are as follows: break for 2 s and stop for 3 s, for a total of 25 min. (8) Centrifuge at 4°C, 12,000 rpm for 20 min to collect the supernatant of the crushing solution, and use a syringe to suck the supernatant and pass through a 0.45 μm PES filter membrane for standby. The single-point mutant nanobody is purified using an AKTA pure protein purification instrument, and the specific steps are as follows: (1) Turn on the machine, and select the System Wash program provided by the machine to flush the ethanol in the machine with deionized water. (2) After the program is completed, install a 5 ml nickel column on the machine, and flush the ethanol in the column with 30 ml of deionized water until the baseline is balanced. The flow rate is set to 5 ml / min, and the upper limit of the pressure is set to 0.5 MPa. (3) Replace the balance buffer to flush and balance the column; the flow rate is set to 3 ml / min. (4) After the baseline is balanced, start loading, and the flow rate is 0.8~1.0 ml / min; after loading is completed, first flush slowly with 10 ml of balance buffer (the flow rate is the same as loading), and then flush at a flow rate of 3 ml / min until the baseline is balanced. (5) Replace the elution buffer to elute, collect the elution peak protein, and set the flow rate to 3 ml / min; after all the proteins are completely eluted, flush the nickel column with deionized water until the ion concentration baseline is zero, and set the flow rate to 5 ml / min. (6) Continue to flush 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 components obtained in the previous step, concentrate to 750 μL at 4°C, 4000 rpm, and pass the concentrated solution through a 0.22 μm PES filter membrane.(2) Use system wash method to clean the internal pipeline of AKTA pure protein purification instrument. (3) After cleaning, install SuperdexTM 75 increase 10 / 300 GL pre-packed column on the instrument, and install a 500 μL sample loop on the instrument, set the flow rate to 0.4 mL / min, and the upper limit of the pressure to 2.8 MPa; (4) Use the Load mode, use 1-2 column volumes of deionized water to clean the column and set the baseline, then change to PBS solution, 1-2 column volumes to balance the column, and at the same time use a syringe to suck 5 mL of PBS solution through the inject hole to clean the sample loop; (5) Use a 1 mL syringe to suck the sample, inject the sample into the sample loop through the inject hole, and change the Load mode to Inject mode; (6) When the detector appears an ultraviolet absorption peak, start collecting, until all the peaks are collected, change the Inject mode back to the Load mode, continue to flush the column and pipeline, and then use deionized water to flush and set the baseline; (7) Use 20 % ethanol to flush the system, 30 mL, then remove the column and store it at 4 °C, turn off the instrument; (8) Perform SDS-PAGE gel electrophoresis experiment to analyze the purified components.

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

[0066] Kinect and affinity program to determine the KD value of the antibody: (1) Use PBS solution to dilute the antibody gradient to 200-0.78125 nM and set the program to load the sample. The chip regeneration solution is 10 mM glycine solution (pH 2.0). (2) After the experiment is completed, disconnect the instrument and import the obtained data into the instrument software for analysis. After data fitting, calculate the affinity constant K. D The results of SPR determination of nanobody affinity constants are shown in Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D and Table 4.

[0067] Table 4 SPR results

[0068] 4. Fluorescence real-time quantitative PCR verification of the thermal stability of nanoantibodies By setting a temperature gradient, the degree of exposure of protein hydroxyl groups is measured, and changes in the protein's spatial structure with temperature are analyzed to assess the thermal stability of the protein. The thermal stability of the nanobody was evaluated using real-time fluorescence quantitative PCR using a Thermo Fisher Scientific Q5 Real-Time PCR System. Three replicates were set for each sample. After PCR completion, the data was imported into the instrument's accompanying software for analysis and calculation of the nanobody's Tm value. Some of the thermal stability results are shown in Table 5.

[0069] Table 5 Thermal stability results

[0070] 5. Verification of the blocking activity of nanobodies against CXCL1 The HCT116 human colon cancer cells were divided into nine groups: 0 nmol / L CXCL1 group, 10 nmol / L CXCL1 group, 10 nmol / L CXCL1+Y1-3F group, 10 nmol / L CXCL1+Y1-2C group, 10 nmol / L CXCL1+Y1-8G group, 10 nmol / L CXCL1+Y1-3E group, 10 nmol / L CXCL1+Y1-1A group, 10 nmol / L CXCL1+Y2-3F group and 10 nmol / L CXCL1+Corydalmine group. The cells were digested according to the cell passage procedure, and blown into a suspension. The cells were counted, and inoculated in the corresponding size culture dishes according to the proportion required by different experiments. After the plates were placed in a 37 ℃, 5% CO2 incubator for culture, the old culture medium was discarded after the cells adhered, and the cells were treated with different concentrations of exogenous CXCL1 (0 or 10 nmol / L) in 5% FBS medium. The antibody and blocker treatment groups were first treated with antibodies and blockers, and then treated with exogenous CXCL1. Then the cells were continued to be cultured in the incubator.

[0071] As shown in Figure 5 compared with the control (0 nmol / L CXCL1) group, the clone formation rate of the 10 nmol / L CXCL1 control group was significantly increased, indicating that CXCL1 had a promoting effect on clone formation. In addition, the clone rate of the positive control 10 nmol / L CXCL1+Corydalmine group was significantly reduced, indicating that blocking the CXCL1 / CXCR2 axis could effectively inhibit the proliferation of HCT116 cells. In addition, compared with the control and 10 nmol / L CXCL1 control groups, the number of clones in the antibody treatment groups was reduced, and the affinity was linearly related to the antibody, indicating that the screened CXCL1 antibodies all had good biological activity.

[0072] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application 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 replacements of some parts. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application. Although the specific embodiments of the present application have been described above, the present application is not limited to the above. Those skilled in the art should understand that various modifications or changes made to the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. An anti-CXCL1 nanobody, characterized in that The nanobody comprises a heavy chain variable region targeting CXCL1, consisting 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.

2. An isolated nucleic acid molecule, characterized in that Encoding the nucleic acid according to claim 1 or its complementary sequence.

3. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that Comprising the recombinant expression vector of claim 3, or comprising 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 comprises the anti-CXCL1 nanobody according to claim 1, and a coupling portion, wherein the coupling portion is divided into a detectable marker, a drug, a toxin, a cytokine, a viral coat protein or a virus-like particle.

6. A conjugate, characterized in that It comprises the anti-CXCL1 nanobody according to claim 1, and a coupling portion; preferably, the coupling portion is selected from a protein tag, a detectable marker, a therapeutic agent or another biologically active polypeptide.

7. A detection kit comprising the anti-CXCL1 Nanobody according to claim 1, and / or the immunoconjugate according to claim 5, and / or the conjugate according to claim 6; the kit can be used to detect the presence or level of CXCL1 in a sample.

8. A pharmaceutical composition, characterized in that It includes the composition of claim 1, the immunoconjugate of claim 5, or the conjugate of claim 6.

9. Use of the Nanobody according to claim 1, the immunoconjugate according to claim 5, the conjugate according to claim 6, the detection kit according to claim 7, or the pharmaceutical composition according to claim 8 in any one or more of the following: (a1) Preparation of products for the detection, monitoring, diagnosis and / or prognosis of tumors, autoimmune diseases or neurological diseases; (a2) Preparation of products for the prevention and / or treatment of tumors, autoimmune diseases or neurological diseases; The tumor, autoimmune disease or neurological disease is specifically manifested by abnormally high expression of CXCL1.

10. Use of the anti-CXCL1 nanobody according to claim 1 in preparing a fusion protein.

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