Cxcl-bpi fusion proteins and uses thereof

Abstract

The application discloses a CXCL-BPI fusion protein which can be used for treating gram-negative bacterial infection, a coding nucleic acid of the CXCL-BPI fusion protein, an expression preparation method, and application of the CXCL-BPI fusion protein to preparation of a pharmaceutical composition for treating gram-negative bacterial infection. The CXCL-BPI fusion protein comprises a human ELR+CXC chemokine and a human BPI N-terminal domain functional fragment, has the functions of combining LPS, directly killing gram-negative bacteria and chemotactic cell migration, and also has the functions of promoting phagocytes to be guided to combine and phagocytize gram-negative bacteria, and the action mechanism can overcome gram-negative bacterial drug resistance.

Description

Invention Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a CXCL-BPI fusion protein for treating Gram-negative bacterial infections, its encoded nucleic acid, a method for expression and preparation, and its use in preparing pharmaceutical compositions for treating Gram-negative bacterial infections. The CXCL-BPI fusion protein contains functional fragments of human ELR+CXC chemokine and the N-terminal domain of human BPI, possessing the dual functions of ELR+CXC chemokine and BPI. It has the functions of binding LPS, directly killing Gram-negative bacteria, and promoting chemotactic cell migration. It also promotes the phagocytic binding and phagocytosis of Gram-negative bacteria by phagocytes, and its mechanism of action can overcome drug resistance in Gram-negative bacteria. Background of the Invention

[0002] Chemokines are a family of small molecular weight cytokines that exhibit chemotactic activity towards cells, especially leukocytes. Chemokine receptors (CKRs) are a superfamily of G protein-coupled receptors (GPCRs) that mediate the function of chemokines. Chemokines mediate the migration of immune cells to sites of infection and inflammation, and activate immune cells to participate in immune responses and inflammatory reactions. Phagocytes such as granulocytes, monocytes / macrophages in higher animals have phagocytic and bactericidal functions and are an important component of the body's non-specific immune function. Chemokines and phagocytes play important roles in the body's immunity against bacterial infections, but they do not possess the specific mechanism of action of opsonization, which promotes the phagocytosis of bacteria, similar to that of specific IgG antibodies. Chemokines are classified into four subfamilies based on the arrangement of the two cysteine ​​residues near their N-terminus: CC, CXC, CX3C, and C. The CXC subfamily is further divided into ELR+CXC chemokines and non-ELR+CXC chemokines based on the presence or absence of an ELR (Glu-Leu-Arg) motif before the first Cys residue. From 1986 to the early 1990s, IL-8, CXCR1, and CXCR2 (also known as IL-8RA and IL-8RB) and their ELR+CXC chemokine ligands were discovered. CXCR1 and CXCR2 are G protein-coupled receptors (GPCRs) primarily expressed on cells such as neutrophils, monocytes / macrophages, and endothelial cells. The ligands of CXCR1 and CXCR2 constitute the ELR+CXC chemokine family. The seven known human ELR+CXC chemokines are Gro-α (also known as CXCL1), Gro-β (CXCL2), Gro-γ (CXCL3), ENA-78 (CXCL5), GCP-2 (CXCL6), NAP-2 (CXCL7), and IL-8 (CXCL8). Their main functions are to primarily chemotactically attract neutrophils (and also have chemotactic effects on monocytes / macrophages) and promote angiogenesis. All seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) mediate signaling by binding to CXCR2, while CXCL6 and CXCL8 also mediate signaling by binding to CXCR1 (all contributing to redundancy in the activation pathway). IL-8 (CXCL8) is a representative member of the CXC subfamily, and its main functions are to chemotactically attract and activate neutrophils, promote neutrophil phagocytosis, and also have some chemotactic and activating effects on monocytes / macrophages.See, for example, [Hughes, et al., FEBS J. (2018) 285 (16): 2944][Bacon, et al., J. Interferon Cytokine Res. (2002) 22 (10): 1067][Bi Huijuan et al., Journal of Immunology (2010) 26 (12): 1091][Baggiolini et al., Adv Immunol. (1993) 55: 97].

[0003] Bactericidal / permeability-increasing protein (BPI) is a cationic antimicrobial protein composed of 456 amino acid residues with a molecular weight of approximately 55 kDa, first discovered by Weiss et al. in human polymorphonuclear neutrophils in 1978. The protein's structure consists of an N-terminal domain and a C-terminal domain linked by a linker. Studies have confirmed that its N-terminal functional fragment, BPI... 1-199 and BPI 1-193 (These are amino acid residues 1-199 of the N-terminal domain and amino acid residues 1-193, truncated by 6 amino acids at the C-terminus, respectively) possess high affinity for binding lipopolysaccharide (LPS) and lipid A of Gram-negative bacteria (GNB or G-bacteria), neutralize endotoxins, and increase the membrane permeability of susceptible GNB, thus directly killing GNB. XOMA has been developing the recombinant human BPI N-terminal functional fragment since the 1990s. rBPI 21 Several clinical trials have been conducted, but because BPI requires a prolonged period to maintain a high concentration for sterilization, while rBPI... 21Due to factors such as short in vivo half-life, large therapeutic dose, and difficulty in maintaining effective therapeutic concentrations in vivo, it has not achieved clinical success and has not been approved by the FDA. See, for example, [Weiss, et al. J. Biol. Chem. (1978) 253: 2664][Gray, et al., J Biol Chem. (1989) 264: 9505][Kleiger, et al., J. Mol. Biol. (2000) 299: 1019][Elsbach, J. Leukoc. Biol. (1998) 64: 14][Levin, et al., Lancet. (2000) 356: 961][Giroir, et al., Crit Care Med (2001) 29(7)(Suppl.): S130].

[0004] Gram-negative bacteria are among the most common pathogens causing infectious diseases. They exhibit strong resistance to commonly used antibiotics and are currently the most concerning drug-resistant pathogens. In February 2017, the WHO released its first list of 12 drug-resistant bacteria posing the greatest threat to human health, nine of which were Gram-negative. More than half of bacterial infections are caused by Gram-negative bacteria, and severe Gram-negative infections can develop into sepsis, leading to endotoxin-induced toxic shock and death; currently, there is still no effective treatment.

[0005] Therefore, the purpose of this invention is to provide a CXCL-BPI fusion protein by utilizing the biological functional characteristics of ELR+CXC chemokine and BPI, which can be used to treat Gram-negative bacterial infections, and its mechanism of action can overcome Gram-negative bacterial resistance. Invention Overview

[0006] The purpose of this invention is to provide a fusion protein containing chemokines and BPI, which has the dual functions of chemokines and BPI. It has the functions of binding to LPS, directly killing Gram-negative bacteria and promoting the migration of chemotactic cells, and also has the function of promoting the phagocytic binding and phagocytosis of Gram-negative bacteria by phagocytes (with the characteristics of specific mechanism of action). Its mechanism of action can overcome drug resistance of Gram-negative bacteria and can be used to treat Gram-negative bacterial infections and to prepare pharmaceutical compositions for treating Gram-negative bacterial infections.

[0007] In one aspect, the present invention provides a fusion protein comprising a chemokine and a BPI, and the nucleic acid encoded therein, which can be used to treat Gram-negative bacterial infections, optionally wherein the nucleic acid is encoding DNA.

[0008] Specifically, in this invention, the chemokine is preferably human ELR+CXC chemokine, and the BPI is preferably a functional fragment of the N-terminal domain of human BPI, providing a CXCL-BPI fusion protein containing human ELR+CXC chemokine and a functional fragment of the N-terminal domain of human BPI.

[0009] In another aspect, the present invention provides the encoding nucleic acid of the CXCL-BPI fusion protein, as well as its efficient expression vector, stable expression host cell and extraction and preparation method thereof, wherein the encoding nucleic acid is encoding DNA.

[0010] In another aspect, the invention provides the use of the CXCL-BPI fusion protein for the treatment of Gram-negative bacterial infections, and / or for the preparation of pharmaceutical compositions for the treatment of Gram-negative bacterial infections. Invention Details

[0011] The objectives and implementation of this invention will be further elaborated below, and those skilled in the art will readily recognize the scope, content, and advantages of this invention. While preferred embodiments are provided, those skilled in the art will recognize that various modifications and variations are also within the scope of this invention. Therefore, the appended claims and any future amendments and variations are within the scope described.

[0012] Chemokines mediate the migration of immune cells to sites of infection and inflammation, activating them to participate in immune responses and inflammatory reactions. Phagocytes such as granulocytes, monocytes / macrophages in higher animals have phagocytic and bactericidal functions and are important components of the body's non-specific immune function. While chemokines and phagocytes play crucial roles in the body's antibacterial immunity, they lack the specific mechanism of action of specific IgG antibodies in promoting bacterial phagocytosis.

[0013] CXCR1 and CXCR2 are G protein-coupled receptors (GPCRs) mainly expressed on cells such as neutrophils, monocytes / macrophages, and endothelial cells. The ligands of CXCR1 and CXCR2 form the ELR+CXC chemokine family, whose main functions are to primarily chemotactically attract neutrophils (and also have chemotactic effects on monocytes / macrophages) and promote angiogenesis. Their biological functions are not species-specific. Among them, IL-8 (CXCL8) is a representative member of the CXC subfamily. Its main functions are to chemotactically attract and activate neutrophils, promote phagocytosis by neutrophils, and also have some chemotactic and activating effects on monocytes / macrophages.

[0014] The N-terminal domain functional fragment of human BPI has the same high affinity as natural human BPI for binding to the lipopolysaccharide (LPS) and lipid A of Gram-negative bacteria GNB, neutralizing endotoxin, and increasing the membrane permeability of susceptible GNB, thus directly killing GNB. However, due to the difficulty in maintaining a high concentration of BPI alone for a long time to kill bacteria, and the difficulty in maintaining an effective therapeutic concentration in vivo, clinical success has been difficult to achieve.

[0015] Therefore, in one aspect, the present invention provides a fusion protein comprising a chemokine and a BPI; specifically, the chemokine is preferably a human ELR+CXC chemokine that functions through CXCR1 and CXCR2 expressed on neutrophils, monocytes / macrophages, and endothelial cells, mediated by these pathways; the BPI is preferably a functional fragment of the N-terminal domain of a human BPI; and a CXCL-BPI fusion protein is provided comprising a human ELR+CXC chemokine and a functional fragment of the N-terminal domain of a human BPI. The human ELR+CXC chemokine is selected from CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 (collectively referred to as CXCL), and the functional fragment of the N-terminal domain of the human BPI is selected from the N-terminal domain of the BPI. 1-233 (The first 233 amino acid residues of the N-terminal domain), BPI 1-199 and BPI 1-193 In the specific implementation plan, CXCL1-BPI, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI, CXCL7-BPI, and CXCL8-BPI fusion proteins (collectively referred to as CXCL-BPI) were designed and constructed; among them, human ELR+CXC chemokine was preferred as the N-terminal domain of the fusion protein, and the N-terminal domain of human BPI was preferred. 1-233 As the C-terminal domain of a fusion protein, it is preferably linked via a linker, and from the N-terminus to the C-terminus, it sequentially comprises CXCL, a linker, and BPI. 1-233 Composed of sequential elements.

[0016] Another aspect of the present invention provides the encoding DNA sequence of the CXCL-BPI fusion protein, as well as its efficient expression vector, stable expression host cell, and extraction and preparation method. Specifically, the present invention designs and optimizes the encoding DNA sequence of the CXCL-BPI fusion protein, which, from the 5' end to the 3' end, sequentially includes a 5' adapter sequence (containing an EcoRI restriction site), a signal peptide coding sequence, a CXCL coding sequence, a linker coding sequence, and a BPI... 1-233The protein consists of a coding sequence and a 3' adapter sequence (containing a TGA stop codon and a Sal I restriction site); a high-efficiency expression vector was constructed, and the CXCL-BPI fusion protein was stably and efficiently expressed in mammalian cells and extracted for preparation.

[0017] This invention confirms that the CXCL-BPI fusion protein possesses the dual functions of ELR+CXC chemokine and BPI, exhibiting the ability to bind LPS, directly kill Gram-negative bacteria, and promote chemotactic cell migration. It also promotes the guided binding and phagocytosis of Gram-negative bacteria by phagocytes (characteristic of a specific mechanism of action), and its mechanism of action can overcome Gram-negative bacterial resistance. Furthermore, the CXCL-BPI fusion protein demonstrates significant bactericidal effects in peripheral blood and peritoneal phagocytes, and significant protective effects against Gram-negative bacterial infections in mice. Therefore, in another aspect, the CXCL-BPI fusion protein can be used to treat Gram-negative bacterial infections and to prepare pharmaceutical compositions for treating Gram-negative bacterial infections.

[0018] Gram-negative bacteria are among the most common pathogens causing infectious diseases, exhibiting strong resistance to commonly used antibiotics and are currently the most concerning drug-resistant pathogens. In February 2017, the WHO released its first list of 12 drug-resistant bacteria posing the greatest threat to human health, nine of which were Gram-negative. More than half of bacterial infections in patients are caused by Gram-negative bacteria, and severe Gram-negative infections can develop into sepsis, leading to endotoxin-induced toxic shock and death; currently, there is no effective treatment. The CXCL-BPI fusion protein described in this invention, used to treat Gram-negative bacterial infections, has significant clinical demand and application prospects.

[0019] Therefore, in one aspect, the present invention provides a CXCL-BPI fusion protein comprising human ELR+CXC chemokine and a functional fragment of the N-terminal domain of human BPI.

[0020] In one embodiment, the human ELR+CXC chemokine in the fusion protein is selected from human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6, human CXCL7, and human CXCL8. Optionally, human CXCL8, human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6, or human CXCL7 respectively comprise the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0021] In another embodiment, the functional fragment of the human BPI N-terminal domain in the fusion protein is selected from human BPI.1-233 Fragments, Human BPI 1-199 Fragments and People BPI 1-193 Fragments, optionally, in which human BPI 1-233 The fragment contains the sequence shown in SEQ ID NO: 10.

[0022] In yet another embodiment, the human ELR+CXC chemokine serves as the N-terminal domain of the fusion protein, and the functional fragment of the human BPI N-terminal domain serves as the C-terminal domain of the fusion protein. The two are optionally connected by a linker, and more optionally, the linker is selected from GPPSSGSGGGSGGG (SEQ ID NO: 8) and GGGSGGGSGGG (SEQ ID NO: 9).

[0023] In another aspect, the present invention provides a nucleic acid encoding the CXCL-BPI fusion protein of the present invention.

[0024] In one embodiment, the nucleic acid comprises, from the 5' end to the 3' end, a 5' end adapter sequence, a signal peptide coding sequence, a human ELR+CXC chemokine coding sequence, an adapter coding sequence, a human BPI N-terminal domain functional fragment coding sequence, and a 3' end adapter sequence. Optionally, the human ELR+CXC chemokine coding sequence comprises the sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, and the human BPI N-terminal domain functional fragment coding sequence comprises the sequence shown in SEQ ID NO: 22.

[0025] In another aspect, the present invention provides the use of the CXCL-BPI fusion protein of the present invention for the treatment of Gram-negative bacterial infections, and / or for the preparation of pharmaceutical compositions for the treatment of Gram-negative bacterial infections.

[0026] In another aspect, the present invention provides an expression vector for expressing the CXCL-BPI fusion protein of the present invention, optionally said expression vector comprising nucleic acid encoding the CXCL-BPI fusion protein of the present invention.

[0027] In one embodiment, the expression vector is selected from the high-efficiency expression vectors pSCm-CXCL1-BPI, pSCm-CXCL2-BPI, pSCm-CXCL3-BPI, pSCm-CXCL5-BPI, pSCm-CXCL6-BPI, pSCm-CXCL7-BPI, and pSCm-CXCL8-BPI.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising the CXCL-BPI fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0029] In another aspect, the present invention provides a host cell comprising an expression vector that stably transfects or transforms a nucleic acid encoding the CXCL-BPI fusion protein of the present invention.

[0030] In another aspect, the present invention provides a method for preparing the CXCL-BPI fusion protein of the present invention, comprising culturing the host cells of the present invention under conditions suitable for the expression of the CXCL-BPI fusion protein, harvesting the expressed CXCL-BPI fusion protein, and optionally further purifying the expressed CXCL-BPI fusion protein.

[0031] In another aspect, the present invention provides a method for treating Gram-negative bacterial infection, comprising: administering a therapeutically effective amount of the CXCL-BPI fusion protein or pharmaceutical composition of the present invention to a subject suffering from a Gram-negative bacterial infection.

[0032] In one embodiment, the method of treating Gram-negative bacterial infection of the present invention further includes administering an antibiotic compound to a subject suffering from Gram-negative bacterial infection before, simultaneously with or after administering a therapeutically effective amount of the CXCL-BPI fusion protein or pharmaceutical composition of the present invention. Brief description of the attached figures

[0033] Figure 1. Extraction and preparation of CXCL-BPI fusion protein. Where: A, SP Typical Fast Flow cation exchange chromatography pattern; B. SDS-PAGE electrophoresis of purified target protein.

[0034] Figure 2 .CXCL-BPI fusion protein binds to endotoxin.

[0035] Figure 3 The CXCL-BPI fusion protein directly kills Gram-negative bacteria.

[0036] Figure 4 .CXCL-BPI fusion protein chemotactic activity in human HL-60 cells.

[0037] Figure 5 CXCL-BPI fusion protein chemotactic on mouse bone marrow neutrophils.

[0038] Figure 6 CXCL-BPI fusion protein chemotactic mouse peritoneal cells.

[0039] Figure 7The CXCL-BPI fusion protein promotes phagocytic guidance and phagocytosis of Gram-negative bacteria. Among them: A / B, human HL-60 cells; C, human peripheral blood leukocytes; D, mouse peripheral blood leukocytes; E, mouse peritoneal phagocytic cells.

[0040] Figure 8 bactericidal activity of CXCL-BPI fusion protein in human / mouse peripheral blood.

[0041] Figure 9 bactericidal effect of CXCL-BPI fusion protein in mouse peripheral blood.

[0042] Figure 10 The bactericidal effect of CXCL-BPI fusion protein in mouse peritoneal phagocytes.

[0043] Figure 11 The protective effect of .CXCL-BPI fusion protein against Gram-negative bacterial infection in mice. A. Mouse infection model (dose); B. Serum; C. Liver; D. Spleen.

[0044] Biological Preservation Information

[0045] This invention relates to Escherichia coli, deposited on September 15, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 25726 and name pSCm-IL8-BPI (in E. coli JM108), located in Beijing, China.

[0046] The technical solutions of the present invention are further described below with reference to embodiments and accompanying drawings, but are not limited to these embodiments. More specifically, Embodiment 1 relates to the CXCL-BPI fusion protein and its encoding DNA sequence, as well as its expression and preparation; Embodiment 2 relates to the biological functions of the CXCL-BPI fusion protein, which has the functions of binding LPS, directly killing Gram-negative bacteria, and promoting chemotactic cell migration, and also has the function of promoting phagocytic cell-guided binding and phagocytosis of Gram-negative bacteria; Embodiment 3 relates to the bactericidal effect of the CXCL-BPI fusion protein in peripheral blood and peritoneal phagocytic cells; Embodiment 4 relates to the protective effect of the CXCL-BPI fusion protein against Gram-negative bacteria infection in mice. The scope, content, and advantages involved in these embodiments are obvious, and various modifications and variations are also within the scope of this description; including, but not limited to, the CXCL-BPI fusion protein and its encoding DNA sequence, as well as other equivalents, isoforms, variants, and analogues of each component element.

[0047] Example 1: CXCL-BPI fusion protein, its encoding DNA sequence, and expression preparation

[0048] 1. CXCL-BPI fusion protein and its encoding DNA sequence

[0049] This invention designs and constructs a CXCL-BPI fusion protein, which, from the N-terminus to the C-terminus, comprises CXCL, a linker, and BPI. 1-233 The sequence elements are composed (as shown in Table 1). This invention designs and optimizes the coding DNA sequence of the CXCL-BPI fusion protein, which, from the 5' end to the 3' end, sequentially includes a 5' adapter sequence (containing an EcoRI restriction site), a signal peptide coding sequence, a CXCL coding sequence, a linker coding sequence, and a BPI coding sequence. 1-233 The sequence consists of a coding sequence and a 3' adapter (containing a TGA stop codon and a Sal I restriction site) (as shown in Table 2).

[0050] Table 1. Composition of CXCL-BPI fusion protein

[0051]

[0052] Table 2. Composition of the encoding DNA sequence of the CXCL-BPI fusion protein

[0053]

[0054]

[0055] The sequences of CXCL1-BPI, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI, CXCL7-BPI, and CXCL8-BPI in the CXCL-BPI fusion protein are shown below:

[0056] CXCL1-BPI:ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSNGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH(SEQ ID NO:24)

[0057] CXCL2-BPI:APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSNGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH(SEQ ID NO:25)

[0058] CXCL3-BPI:ASVVTELRCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKACLNPASPMVQKIIEKILNKGSTNGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH(SEQ ID NO:26)

[0059] CXCL5-BPI:LRELRCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDPEAPFLKKVIQKILDGGNKENGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH(SEQ ID NO:27)

[0060] CXCL6 - BPI: VLTELRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDPEAPFLKKVIQKILDSGNKKNGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH (SEQ ID NO: 28)

[0061] CXCL7 - BPI: AELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESADGGGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH (SEQ ID NO: 29), and

[0062] CXCL8-BPI: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAEGPPSGSGGGSGGGVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVP NVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH(SEQ ID NO: 30).

[0063] 2. High-efficiency expression vector for CXCL-BPI fusion protein

[0064] The encoding DNA sequence of the CXCL-BPI fusion protein, designed and optimized by a commercial gene synthesis service (Nanjing GenScript), was constructed using conventional molecular cloning techniques at the EcoRI / SalI double restriction site of the pSCm-IL8-BPI eukaryotic expression vector (constructed by the inventors and deposited on September 15, 2022, at the China General Microbiological Culture Collection Center, accession number CGMCC NO.: 25726) (transformed into E. coli JM108). The correctly constructed high-efficiency CXCL-BPI expression vectors pSCm-CXCL1-BPI, pSCm-CXCL2-BPI, pSCm-CXCL3-BPI, pSCm-CXCL5-BPI, pSCm-CXCL6-BPI, pSCm-CXCL7-BPI, and pSCm-CXCL8-BPI (collectively referred to as pSCm-CXCL-BPI) were obtained.

[0065] 3. Stable and efficient expression and extraction preparation of CXCL-BPI fusion protein

[0066] Use CHO CD1 serum-free culture medium (with 1×L-alanyl-glutamine solution) (Shanghai Yuanpei) was used to acclimate CHO-K1 cells (ATCC CCL-61) at a concentration of 3–5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 / mL and cultured for 24 h (37℃, 5% CO2, 130 rpm). 400 μL of cell suspension (1 × 10⁻⁶ cells / mL) was collected by centrifugation. 7After mixing 6 μg of pSCm-CXCL-BPI plasmid (1 μg / μL) with the cells / mL, transfer the mixture into a 0.8 mL electroporation cuvette and follow the BTX protocol. The 830 electroporation system manual states: 360V, 7ms twice (1s interval), transfer cells to two 10cm cell culture dishes (10mL / dish) and incubate statically for 24h; then replace with selective medium (CHO containing 30μM MSX). CD1), press 2×10 3 Cells were seeded into 96-well plates, with fluid replenishment every 5–7 days. Once the clones reached 1 / 3 of the well area, the high-efficiency expression level of the target protein in the supernatant was continuously evaluated (ELISA and SDS-PAGE). High-efficiency expression clones were progressively expanded to 125 mL shake flasks, with protein and cell quality assessments used for selection. Ultimately, 5–10 stable high-expression clones (expression level 20–60 pcd) were retained for each CXCL-BPI fusion protein.

[0067] The cells that efficiently express the CXCL-BPI fusion protein obtained above are divided into 3×10 cells. 5 Cells / mL were seeded in cell shake flasks (Nalgene) TM PETG, 250 mL) in (culture medium containing 30 μM MSX CHO) CD1), and add an appropriate amount of SP. Fast Flow co-culture (37℃, 5% CO2, 130 rpm) for 8–10 days to capture the target protein; SP was collected. Fast Flow was used for column packing and purification by liquid chromatography. Elution was performed using a salt concentration gradient of 3 mM citrate-13.6 mM phosphate buffer (pH 6.4) containing 0.10, 0.45, and 1.0 M NaCl. Typical peaks of the target protein fraction eluted with 1.0 M NaCl were collected (e.g., peaks of 0.10, 0.45, and 1.0 M NaCl). Figure 1A As shown), replace the protein preservation solution (3mM citrate-13.6mM phosphate buffer containing 0.5M NaCl, pH 6.4) and store at -30℃ for later use.

[0068] The results of SDS-PAGE electrophoresis of the purified target protein showed that: Figure 1B As shown, each CXCL-BPI fusion protein band is clear (high purity) and its position matches the expected molecular weight.

[0069] Example 2: Biological functions of the CXCL-BPI fusion protein

[0070] 1. Combined with endotoxins

[0071] Take 120 μL of CXCL-BPI fusion protein of different concentrations (diluted with endotoxin-free PBS, with a blank control) and 120 μL of endotoxin (2 EU / mL, diluted with water for endotoxin testing), add them to an endotoxin-free glass tube, vortex to mix for 30 s, and incubate at 37°C for 1 h; after vortexing for another 30 s, take 100 μL / well of the mixture into an endotoxin-free 96-well plate and operate according to the instructions for the microplate quantitative chromogenic matrix method (Xiamen Limulus Amebocyte Lysate Reagent Biotechnology Co., Ltd., EC64405).

[0072] The results show that: Figure 2 As shown, the CXCL-BPI fusion proteins can all neutralize (bind) LPS, and this is positively dose-dependent.

[0073] 2. Directly kills Gram-negative bacteria

[0074] Take 50 μL of E. coli BL21(DE3) / pBR322(amp) R and tet R )Bacterial suspension (1×10 4 Mix 50 μL of CXCL-BPI fusion protein (CFU / mL) with 50 μL of different concentrations (both dilution and control were performed using physiological saline), incubate at 37°C for 70 min, and then take 50 μL of each for pour counting.

[0075] The results show that: Figure 3 As shown in Figure A, the CXCL8-BPI fusion protein can directly kill Gram-negative bacteria in a dose-dependent manner; furthermore, as... Figure 3 As shown in B, CXCL1-BPI, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI and CXCL7-BPI can all directly kill Gram-negative bacteria.

[0076] 3. Chemotactic cell migration

[0077] 3.1 Chemotaxis of human HL-60 cells

[0078] Add 600 μL / well of CXCL-BPI fusion protein at different concentrations (IMDM dilution) to the lower chamber of a Transwell (Corning, 3422) apparatus, and add 1.0 × 10⁻⁶ μL / well to the upper chamber. 5 Cells were cultured in 100 μL / well HL-60 cell suspension (without / with 1.25% DMSO induction) at 37℃ and 8% CO2 for approximately 5 h. The chambers were then removed, and cell migration was observed under a microscope. Observation method (the same applies below): Five regions were randomly selected from each well for photographing and counting (selection principle: top left, top right, center, bottom left, bottom right). The chemotaxis index CI = number of cells chemotactically attracted to the test sample solution / number of cells chemotactically attracted to the negative control solution.

[0079] The results show that: Figure 4 As shown in Figure A, the CXCL8-BPI fusion protein exhibits significant chemotactic activity towards the migration of undifferentiated human HL-60 cells (promyelocytes) in a dose-dependent manner; furthermore, as... Figure 4 As shown in B, the CXCL-BPI fusion protein exhibited significant chemotactic activity towards the migration of DMSO-induced differentiated human HL-60 cells (neutrophil-like cells) at their respective optimal protein concentrations.

[0080] 3.2 Chemotaxis of mouse bone marrow neutrophils

[0081] Mice were euthanized by vertebral dislocation and soaked in 75% ethanol for 5–10 min. The tibia and femur were separated, washed in 5 mL PBS, and further muscle tissue was removed. Both ends of the tibia and femur were cut off to expose the medullary cavity. The medullary cavity was rinsed in 8 mL PBS, thoroughly dispersed, and filtered through a 70 μm nylon mesh. Cells were collected by centrifugation at 3000 rpm for 4 min, resuspended in 3 mL IMDM, and neutrophils were prepared by Percoll gradient density centrifugation. The cells were resuspended in 1.3 mL IMDM for later use. The chemotaxis experiment was then performed using the same method as HL-60 cells, except that the culture time at 37℃ and 8% CO2 was adjusted from 5 h to 2 h.

[0082] The results show that: Figure 5 As shown in A, 5B, and 5C, the CXCL1-BPI, CXCL2-BPI, and CXCL8-BPI fusion proteins all exhibited significant chemotactic activity against mouse bone marrow neutrophils, showing a dose-dependent relationship; furthermore, as Figure 5 As shown in Figure D, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI, and CXCL7-BPI all exhibited significant chemotactic activity against mouse bone marrow neutrophils at a protein concentration of 25 μg / mL.

[0083] 3.3 Chemotaxis of mouse peritoneal cells

[0084] Mice were euthanized by vertebral dislocation and immersed in 75% ethanol for 5 minutes. The fur on the surface of the abdomen was cut open, keeping the peritoneum intact. 4-5 mL of IMDM per mouse was injected into the peritoneal cavity of the mouse, and the peritoneal fluid was gently massaged for 5 minutes. The peritoneal fluid was aspirated into a 50 mL centrifuge tube, and this operation was repeated once. The cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the mouse peritoneal cells (containing a large number of phagocytes) were resuspended in 1.3 mL of IMDM for later use. The experimental method of chemotaxis of HL-60 cells was then carried out, except that the culture time at 37℃ and 8% CO2 was adjusted from 5 h to 2-2.5 h.

[0085] The results show that: Figure 6As shown in A and 6B, both CXCL1-BPI and CXCL8-BPI fusion proteins exhibited significant chemotactic activity against mouse peritoneal cells, showing a dose-dependent relationship; furthermore, as Figure 6 As shown in C, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI and CXCL7-BPI all exhibited significant chemotactic function in mouse peritoneal cells at a protein concentration of 25 μg / mL.

[0086] 4. Promotes phagocytic guidance and phagocytosis of Gram-negative bacteria

[0087] 4.1 Human HL-60 cells

[0088] HL-60 cells (induced to differentiate into neutrophils using 1.25% DMSO) were stained with DiI (Beyotime, C1036) for 20 min, washed twice with HBSS, and resuspended in IMDM to a final volume of 5 × 10⁻⁶. 5 Add 400 μL of cells / mL to a 24-well plate, then add 30 μL of 1×10⁻⁶ cells / well. 8 CFU / mL E.coli BL21(DE3) / pET28a-EGFP(kan R Bacterial suspension (with EGFP induced by IPTG as a green fluorescent marker; the same below) and different concentrations of CXCL-BPI fusion protein (both dilution and control were performed using protein dilution buffer) were mixed, incubated at 37°C for 1.5 h, washed twice with HBSS, transferred to new 24-well plates, and observed under an inverted fluorescence microscope; observation method (the same below): DiI red fluorescent markers cells, EGFP green fluorescent markers E. coli BL21(DE3) / pET28a-EGFP, and the two marker images were superimposed (DiI+EGFP), with arrows indicating binding and phagocytosis (EGFP green fluorescence was observed in the cell membrane and intracellular space).

[0089] The results show that: Figure 7 As shown in Figure A, the CXCL8-BPI fusion protein significantly promoted the guided binding and phagocytosis of Gram-negative bacteria in human HL-60 cells (neutrophil-like cells) in a dose-dependent manner; furthermore, as Figure 7 As shown in B, the CXCL-BPI fusion protein significantly promoted the directed binding and phagocytosis of Gram-negative bacteria in human HL-60 cells (neutrophil-like cells) at a protein concentration of 20 μg / mL.

[0090] 4.2 Human peripheral blood leukocytes

[0091] Peripheral blood was collected from individuals, anticoagulated with 0.4% sodium citrate, and erythrocytes were lysed using erythrocyte lysis buffer (Solarbio, R1010). After DiI membrane staining for 45 min, cells were evenly divided according to the experimental group design (approximately 1×10⁻⁶ cells per group).6 Cells / groups were collected by centrifugation at 450g for 5 min and set aside for later use; 100 μL of E. coli BL21(DE3) / pET28a-EGFP bacterial suspension was washed with PBS and prepared to 2.5×10⁻⁶ cells / group. 8 CFU / mL bacterial suspension (with an equal volume of PBS as a negative control) was mixed with 100 μL of CXCL8-BPI fusion protein of different concentrations (with an equal volume of protein dilution as both negative and positive controls), and incubated at 37°C for 20 min. The bacterial and protein suspension was resuspended in the cells to be used, added to a 96-well plate, and incubated at 37°C and 200 rpm for 60 min. The cells were centrifuged at 450 g for 1 min and the supernatant was discarded. The cells were washed once with PBS. The cells were fixed with 4% cell fixative (Solarbio, P1110) for 10 min. After centrifugation and washing in the same manner, the cells were resuspended with an appropriate amount of anti-fluorescence quencher, spotted, mounted, and observed under a fluorescence microscope.

[0092] The results show that: Figure 7 As shown in C, the preferred CXCL8-BPI fusion protein significantly promotes the directed binding and phagocytosis of Gram-negative bacteria by human peripheral blood phagocytes (mainly neutrophils, followed by monocytes), and this effect is dose-dependent.

[0093] 4.3 Mouse peripheral blood leukocytes

[0094] Blood was collected from the jaw of mice, anticoagulated with 0.4% sodium citrate, and erythrocytes were lysed with erythrocyte lysis buffer. The experiment was performed using the same method as in 4.1 human HL-60 cells and with 20 μg / mL CXCL-BPI fusion protein.

[0095] The results show that: Figure 7 As shown in Figure D, the CXCL-BPI fusion protein significantly promoted the guided binding and phagocytosis of Gram-negative bacteria by peripheral blood phagocytes (mainly neutrophils, followed by monocytes) in mice.

[0096] 4.4 Mouse peritoneal phagocytes

[0097] Mouse peritoneal cells were prepared according to the experimental method in 3.3. After resuspending in an appropriate amount of DMEM-H, the cells were seeded at 100 μL / well on 24-well plates and cultured at 37°C and 8% CO2 to allow the cells to adhere. After DiI membrane staining, the experiments were conducted according to the experimental method for human peripheral blood leukocytes in 4.2, using different concentrations of the preferred CXCL8-BPI fusion protein. The difference was that the peritoneal phagocytes were in an adherent state on the plate, and centrifugation was not required during the operation.

[0098] The results show that: Figure 7As shown in E, the preferred CXCL8-BPI fusion protein significantly promotes the directed binding and phagocytosis of Gram-negative bacteria by mouse peritoneal phagocytes (including macrophages and neutrophils) in a dose-dependent manner.

[0099] Example 3: Bactericidal effect of CXCL-BPI fusion protein in peripheral blood and peritoneal phagocytes

[0100] Given that human peripheral blood (from healthy volunteers) exhibits strong resistance to E. coli BL21(DE3) (such as serotype reaction and phagocytic clearance), Acinetobacter baumannii was selected for the human peripheral blood bactericidal experiment in this embodiment, while Acinetobacter baumannii and E. coli BL21(DE3) could be selected for the mouse peripheral blood and mouse peritoneal phagocyte bactericidal experiments.

[0101] 1. Human / mouse peripheral blood bactericidal experiment

[0102] Take 100 μL of Acinetobacter baumannii (ATCC BAA-1605, multidrug-resistant) bacterial suspension (2 × 10⁻⁶). 4 The following were mixed: 100 μL of CXCL-BPI fusion protein at different concentrations (diluted in physiological saline), 180 μL of physiological saline, and 20 μL of human / mouse peripheral blood (0.4% sodium citrate anticoagulation). The experiment included a heat-treated human / mouse peripheral blood control group (heat treatment: 56℃ water bath for 30 min to inhibit or destroy phagocytes, complement and other related biological activities), and incubation at 37℃ for 1 h; 50 μL of each was taken for pour point counting.

[0103] The results show that: Figure 8 As shown in A / 8C, within a relatively low concentration range, the preferred CXCL8-BPI fusion protein exhibited significantly higher bactericidal effects in human / mouse peripheral blood than the heat-treated human / mouse peripheral blood control group, and this difference was negatively correlated with concentration (i.e., the lower the concentration, the more significant the difference), suggesting its role in promoting the guided binding and phagocytosis of Gram-negative bacteria by peripheral blood phagocytes; furthermore, as Figure 8 As shown in B / 8D, CXCL1-BPI, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI and CXCL7-BPI, at their respective optimal protein concentrations, all exhibited significantly higher bactericidal effects in human / mouse peripheral blood than the heat-treated human / mouse peripheral blood control group.

[0104] 2. Mouse peripheral blood bactericidal experiment

[0105] The experiment was conducted according to the method described in section 1 above. 100 μL of LE.coli BL21(DE3) / pBR322 bacterial suspension (1×10⁻⁶) was taken. 4100 μL of CXCL8-BPI fusion protein at different concentrations (both dilution and control were performed using protein dilution buffer) and 40 μL of mouse peripheral blood (0.4% sodium citrate anticoagulation) were mixed and incubated at 37°C for 1.5 h; 100 μL of each was then used for the spread plate method for counting.

[0106] The results show that: Figure 9 As shown in Figure A, the preferred CXCL8-BPI fusion protein exhibits significant bactericidal effects in mouse peripheral blood, showing a dose-dependent relationship; furthermore, as... Figure 9 As shown in B, the CXCL1-BPI, CXCL2-BPI, CXCL3-BPI, CXCL5-BPI, CXCL6-BPI and CXCL7-BPI fusion proteins all exhibited significant bactericidal effects in mouse peripheral blood.

[0107] 3. Mouse peritoneal phagocyte bactericidal experiment

[0108] Mouse peritoneal cells were prepared according to the experimental method in Example 2, 3.3. An appropriate amount of IMDM was resuspended, and 100 μL / well was seeded into 96-well plates. The plates were incubated statically at 37°C and 8% CO2 for approximately 4 hours to allow cell adhesion and confluence to reach about 80%. An IMDM (cell-free group) was set up as a control. Cells were washed once with 200 μL / well of physiological saline. 1×10⁻⁶ cells were then collected. 4 CFU / mL E. coli BL21(DE3) / pBR322 bacterial suspension was mixed with equal volumes of different concentrations of the preferred CXCL8-BPI fusion protein (both dilutions and controls were performed using physiological saline). After incubation at 37°C for 10 min, 100 μL was added to each well of the aforementioned cell culture medium. The 96-well plate was then incubated at 37°C for 60 min, and 50 μL of each well was collected for pour counting. Furthermore, the experiment was repeated as above, with CHO-DG44 cells (non-phagocytic cell group) used as a control.

[0109] The results show that: Figure 10 As shown in A and 10B, within a relatively low concentration range, the bactericidal effect of the preferred CXCL8-BPI fusion protein in mouse peritoneal phagocytes (MPPs) was significantly higher than that in the cell-free control group and the CHO-DG44 cell control group. Moreover, this difference was negatively correlated with the concentration (i.e., the lower the concentration, the more significant the difference), indicating its role in promoting the directed binding and phagocytosis of Gram-negative bacteria by peritoneal phagocytes.

[0110] Example 4: Protective effect of CXCL-BPI fusion protein against Gram-negative bacteria infection in mice

[0111] 1. Mouse model of Gram-negative bacteria infection

[0112] Mouse infection model (dosage): E. coli BL21(DE3) / pBR322 was diluted with PBS to prepare bacterial suspensions of different concentrations. 6-8 week old mice were randomly divided into groups (n=5 per group) and intraperitoneally injected with the bacterial suspension (0.25 mL / mouse). At 3, 6, 9, 12, and 24 hours, one mouse from each group was collected for ocular blood collection. After standing for 40 min, the blood was centrifuged at 1000 rpm for 10 min. Serum was collected and diluted 10-fold with physiological saline. 50 μL of serum was collected for pour point counting (repeated twice, the same below). The dynamic changes in serum bacterial count under different intraperitoneal injection conditions were statistically observed. Simultaneously, the dynamic changes in coat color, activity, and diarrhea were observed and recorded. The injection dose that resulted in suitable serum bacterial counts and obvious infection symptoms in the mice was determined as the subsequent in vivo experimental dose.

[0113] The results show (e.g.) Figure 11 A), 1×10 8 CFU is only the appropriate dose for in vivo experiments in a mouse model of intraperitoneal infection.

[0114] 2. Protective effect of CXCL-BPI fusion protein against Gram-negative bacterial infection in mice.

[0115] The CXCL8-BPI fusion protein was preferred for this experiment. Randomized 6-8 week old mice (BALB / c) were intraperitoneally injected with 1×10⁻⁶ protein. 8 E. coli BL21(DE3) / pBR322 bacterial suspension (CFU / 0.25 mL / mouse) was used for infection challenge. Ten minutes later, mice were intraperitoneally injected with the preferred CXCL8-BPI fusion protein (0.3 mg / 0.25 mL / mouse; the control group received the corresponding buffer solution). At 2, 4, 6, 8, and 10 hours, 6–7 mice from each group were collected for the following procedures: 1) Blood was collected from the eyeballs, allowed to stand for 40 minutes, centrifuged at 1000 rpm for 10 minutes, and the serum was diluted 10-fold with physiological saline to prepare serum samples. 50 μL of each sample was collected for pour point counting; 2) Organs (liver and spleen) were separated, rinsed with an appropriate amount of sterile physiological saline, ground, resuspended in 3 mL of sterile physiological saline, filtered through a 70 μm sieve, and homogenized. 50 μL of each homogenate was collected for pour point counting. The dynamic changes in bacterial count in the serum and organs of mice in each group were statistically observed, and the dynamic changes in their activity, coat color, diarrhea, and other conditions were also observed and recorded.

[0116] The results show that: Figure 11As shown in B, C, D and Table 3, the preferred CXCL8-BPI fusion protein has a significant protective effect against in vivo infection in mice. The bacterial count in the serum and organs (liver and spleen) of the experimental group mice was significantly lower than that in the control group. At the same time, the condition of the experimental group mice (significantly better, with almost no obvious diarrhea) was significantly better than that of the control group (severely reduced activity, ruffled fur, and diarrhea in most cases within 4-8 hours, and these symptoms were mostly relieved after 10 hours).

[0117] Table 3. Protective effect of CXCL8-BPI against E. coli BL21(DE3) / pBR322-infected mice

[0118]

Claims

1. A CXCL-BPI fusion protein comprising a human ELR+CXC chemokine and a functional fragment of a human BPI N-terminal domain, wherein the human ELR+CXC chemokine is selected from human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6, human CXCL7, and human CXCL8, wherein human CXCL8, human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6, or human CXCL7 are sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively, and wherein the functional fragment of the human BPI N-terminal domain is a human BPI 1-233 Fragment, in which human BPI 1-233 The fragment is the sequence shown in SEQ ID NO:

10. The human ELR+CXC chemokine serves as the N-terminal domain of the fusion protein, and the functional fragment of the human BPI N-terminal domain serves as the C-terminal domain of the fusion protein. The human ELR+CXC chemokine and the human BPI N-terminal domain functional segment are connected by a connector.

2. The CXCL-BPI fusion protein of claim 1, wherein the linker is selected from GPPSSGSGGGSGGG (SEQ ID NO: 8) and GGGSGGGSGGG (SEQ ID NO: 9).

3. A nucleic acid encoding the CXCL-BPI fusion protein according to any one of claims 1-2.

4. The nucleic acid of claim 3, wherein from the 5' end to the 3' end, it sequentially comprises a 5' end adapter sequence, a signal peptide coding sequence, a human ELR+CXC chemokine coding sequence, an adapter coding sequence, a human BPI N-terminal domain functional fragment coding sequence, and a 3' end adapter sequence.

5. The nucleic acid of claim 3, wherein the coding sequences for human ELR+CXC chemokine are the sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, and the coding sequence for the functional fragment of the N-terminal domain of human BPI is the sequence shown in SEQ ID NO:

22.

6. Use of the CXCL-BPI fusion protein according to any one of claims 1-2 for the preparation of pharmaceutical compositions for treating Gram-negative bacterial infections.

7. An expression vector for expressing the CXCL-BPI fusion protein according to any one of claims 1-2.

8. The expression vector of claim 7, comprising nucleic acid encoding the CXCL-BPI fusion protein of any one of claims 1-2.

9. The expression vector of claim 8, wherein the expression vector is selected from the high-efficiency expression vectors pSCm-CXCL1-BPI, pSCm-CXCL2-BPI, pSCm-CXCL3-BPI, pSCm-CXCL5-BPI, pSCm-CXCL6-BPI, pSCm-CXCL7-BPI and pSCm-CXCL8-BPI.

10. A pharmaceutical composition comprising the CXCL-BPI fusion protein of any one of claims 1-2 and a pharmaceutically acceptable carrier.

11. A host cell comprising an expression vector, said expression vector being stably transfected or transformed with nucleic acid encoding the CXCL-BPI fusion protein of any one of claims 1-2.

12. A method for preparing the CXCL-BPI fusion protein according to any one of claims 1-2, comprising culturing the host cells of claim 11 under conditions suitable for CXCL-BPI fusion protein expression, harvesting the expressed CXCL-BPI fusion protein, and optionally further purifying the expressed CXCL-BPI fusion protein.

13. Use of the CXCL-BPI fusion protein of any one of claims 1-2 or the pharmaceutical composition of claim 10 in the preparation of a medicament, wherein the medicament is used to treat Gram-negative bacterial infections, the method comprising: A therapeutically effective amount of the CXCL-BPI fusion protein of any one of claims 1-2 or the pharmaceutical composition of claim 10 is administered to a subject suffering from a Gram-negative bacterial infection.

14. The use of claim 13, wherein the method further comprises administering an antibiotic compound to a subject suffering from a Gram-negative bacterial infection before, simultaneously with or after administering a therapeutically effective amount of the CXCL-BPI fusion protein of any one of claims 1-2 or the pharmaceutical composition of claim 10.

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