Application of hypoxia-induced lipid droplet-associated protein (HILPDA) in the prevention or treatment of hyperlipidemia-related diseases.

Abstract

This invention relates to the use of hypoxia-induced lipid droplet-associated protein (HILPDA) in the prevention or treatment of hyperlipidemia-related diseases. More specifically, it relates to the use of HILPDA in the preparation of medicaments for reducing the levels of apolipoproteins and lipids in the blood, and in the preparation of medicaments for the prevention or treatment of hyperlipidemia-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the use of hypoxia-induced lipid droplet-related proteins in the preparation of drugs for the prevention or treatment of hyperlipidemia-related diseases. Background Technology

[0002] Hyperlipidemia is a very common metabolic disease in modern society, mainly characterized by abnormally high levels of cholesterol, triglycerides, and apolipoproteins in the blood. Surveys show that in my country, over 13% of men and over 23% of women over 60 years of age have hyperlipidemia. Long-term hyperlipidemia can easily lead to atherosclerosis and related cardiovascular diseases, hypertension, hyperglycemia, hyperuricemia, and other complications.

[0003] Currently, statins are the most commonly used lipid-lowering drugs in clinical practice. Their main mechanism of action is to inhibit key enzymes related to intracellular cholesterol synthesis, thereby reducing the amount of cholesterol released into the bloodstream and lowering blood lipid levels. Although statins are widely used clinically, they also have significant limitations, including limited effectiveness in lowering triglycerides and apolipoproteins, and a higher risk of causing side effects such as liver damage and myalgia. Therefore, seeking lipid-lowering drugs that can specifically lower triglycerides and apolipoproteins is of significant clinical importance.

[0004] Hypoxia-induced lipid droplet-associated protein (HILPDA) is a target protein (encoded by the HILPDA gene) regulated by hypoxia-inducible factor 1 / 2 (HIF1 / 2) or peroxisome proliferator-activated receptor (PPAR). It is primarily located in lipid droplets within cells, promoting lipid accumulation. HILPDA consists of 63 amino acids in humans and 64 amino acids in mice.

[0005] There was limited evidence previously that HILPDA, as a small secreted protein, was highly expressed in the blood of patients with clear cell renal cell carcinoma, but this conclusion was subsequently refuted by another research team, so whether it is a secreted protein remains controversial.

[0006] Studies have found that knocking out HILPDA in a mouse model lacking apolipoprotein APOE can reduce the formation of foam cells (characteristic cells in atherosclerosis) derived from macrophages with excessive lipid accumulation, thus mitigating the progression of atherosclerosis. These findings reveal that HILPDA promotes the progression of atherosclerosis in specific APOE-deficient mouse models. Current research is based on the biological functions of HILPDA through interactions with previously known intracellular proteins.

[0007] There are currently no reports of HILPDA reducing the levels of apolipoproteins and lipids in the blood. Summary of the Invention

[0008] Through research, the inventors discovered that hypoxia-induced lipid droplet-associated protein (HILPDA) can indeed act as a secretory protein to effectively promote the clearance of apolipoproteins and lipid particles in serum, such as Apolipoprotein E (ApoE) lipid particles, by various cells cultured in vitro, thereby reducing the content of apolipoproteins (including but not limited to ApoE) and lipid particles in the blood.

[0009] Therefore, in one aspect, the present invention provides the use of hypoxia-induced lipid droplet-associated protein (HILPDA) in the preparation of a medicament for reducing the levels of apolipoproteins and lipids in the blood.

[0010] On the other hand, the present invention provides the use of hypoxia-induced lipid droplet-associated protein (HILPDA) in the preparation of medicaments for the prevention or treatment of hyperlipidemia-related diseases.

[0011] In this invention, the hyperlipidemia-related diseases refer to hyperlipidemia and its complications, namely, excessively high total cholesterol levels in fasting venous serum, equal to or exceeding 6.2 mmol / L; or / and excessively high serum triglyceride levels, equal to or exceeding 2.3 mmol / L; or / and excessively high low-density lipoprotein cholesterol (LDL-cholesterol) levels, equal to or exceeding 4.1 mmol / L; or / and decreased serum high-density lipoprotein cholesterol (HDL-cholesterol) levels, below 1.0 mmol / L, and their derived pathological symptoms, including but not limited to hyperlipidemia, atherosclerosis caused by long-term hyperlipidemia, and complications such as cardiovascular disease, hypertension, hyperglycemia, and hyperuricemia caused by long-term hyperlipidemia.

[0012] In one embodiment, HILPDA may be derived from human (Homo sapiens), chimpanzee (Pantroglodytes) or gorilla (Gorilla), with an amino acid sequence as shown in SEQ ID NO:1 (MKHVLNLYLLGVVLTLLSIFVRVMESLEGLLESPSPGTSWTTRSQLANTEPTKGLPDHPSRSM).

[0013] In another embodiment, HILPDA may be mouse (Mus musculus) HILPDA with an amino acid sequence as shown in SEQ ID NO:2 (MKFMLNLYVLGIMLTLLSIFVRVMESLGGLLESPLPGSSWITRGQLANTQPPKGLPDHPSRGVQ).

[0014] In another embodiment, HILPDA may be HILPDA from the green monkey (Chlorocebus sabaeus), with an amino acid sequence as shown in SEQ ID NO:3 (MKHMLNLYLLGVVLTLLSIFVRVMESLEGLLENPSPGTSWTTRSQLANTEPTKGLPDHPSRSI).

[0015] In another embodiment, HILPDA may be derived from the Qinling golden snub-nosed monkey (Rhinopithecus roxellana), with an amino acid sequence as shown in SEQ ID NO:4 (MKHVLNLYLLGVVLTLLSIFVRVMESLEGLLENPLPGTSWTTRSQLTNTEPTKGLPDHPSRSM).

[0016] In another embodiment, HILPDA may be derived from the cynomolgus macaque (Macaca fascicularis), with an amino acid sequence as shown in SEQ ID NO:5 (MKHMLNLYLLGVVLTLLSIFVRVMESLEGLLENPSPGTSWTTRSQLANTEPTKGLPDHPSRSM).

[0017] In another embodiment, HILPDA may be HILPDA derived from domestic pigs (Sus scrofa), with an amino acid sequence as shown in SEQ ID NO:6 (MKQVLQLYLLGVVLTLLSVFVRLMETLGGLLESPSPGSFWTTRGQLANTEAKSLPEHPSRGV).

[0018] In another embodiment, HILPDA may be HILPDA derived from dogs (Canis familiaris), with an amino acid sequence as shown in SEQ ID NO:7 (MKPMLNLYLLGVVLTLLSIFVRLMESLGGILESPLLGSSWTTRGHTASAEPPRAFQTIHPERCDKTSLHQL).

[0019] In another embodiment, HILPDA may be HILPDA derived from goat (Capra hircus) with an amino acid sequence as shown in SEQ ID NO:8 (MKHVLNLYLLGGVLTLLSIFVRLMESLEGLLESPSPGSSWATRGQLANTEPPRAFQTIHPEGCDKTSLHRL).

[0020] In another embodiment, HILPDA may be HILPDA from horse (Equus caballus) with an amino acid sequence as shown in SEQ ID NO:9 (MKHILNLYLLGVVLTLLSIFVRLMESLEGLLESPSPGSSWTTRSQLVSTR PPRAFQ TIHP EGCDKTSVHRL).

[0021] In another embodiment, HILPDA may be HILPDA derived from bovine taurus, with an amino acid sequence as shown in SEQ ID NO:10 (MKHMLNLYLLGGVMTLLSIFIGLMESLEACWRACLLGAPGPPEVSLPTQSPQGSSRPSIQRGVIRPPSTVYNLGTVSKIPPTLPVPGQASSTSVLSEN).

[0022] In another embodiment, HILPDA may be derived from the rabbit (Oryctolagus cuniculus), with an amino acid sequence as shown in SEQ ID NO:11 (MKHVLNLYVLGVVLALLSIFVRVMESLESLVESPSAGNSWSTRGQLAGAEPPKGLPDPPSRGMR).

[0023] In one embodiment, the drug is used to treat humans or other mammals. Other mammals refer to mammals other than humans, such as apes, monkeys, pigs, dogs, cattle, sheep, horses, donkeys, rabbits, etc.

[0024] In some implementations, HILPDA derived from humans can be used to treat humans or other mammals.

[0025] In some implementations, HILPDA derived from other mammals may be preferred for treating domestic pets of closely related species of the corresponding mammal (e.g., without reproductive isolation). For example, HILPDA derived from dogs may be used to treat dogs or wolves.

[0026] In this invention, preferably, one or more amino acids constituting HILPDA may be D-form amino acids.

[0027] In this invention, preferably, HILPDA can be further modified.

[0028] In this invention, HILPDA can be modified using various techniques known in the art, as long as such modification does not significantly degrade HILPDA's ability to effectively promote the absorption of apolipoproteins and lipid particles in serum. There are many types of peptide modifications, which can be categorized according to the modification site, such as N-terminal modification, C-terminal modification, side chain modification, amino acid modification, and backbone modification. Peptide modification can effectively alter the physicochemical properties of peptide compounds, increase water solubility, prolong in vivo action time, change their biodistribution, eliminate immunogenicity, and reduce toxic side effects.

[0029] For example, modification methods include, but are not limited to, modification by connecting stearic acid to the end (e.g., N-terminus), aminoation modification, acetylation modification, biotinylation modification, fluorescent labeling modification, polyethylene glycol modification, isoprene modification, cardamomylation and palmitoylation modification, phosphorylation modification, glycosylation modification, peptide conjugate modification, and specific amino acid modification.

[0030] The inventors have discovered a novel use for hypoxia-induced lipid droplet-associated protein (HILPDA) through research. Since this naturally occurring small protein or polypeptide originates from the human body, it should exhibit low toxicity and lack immunogenicity when administered to patients, making it suitable for clinical translation and widespread application. Therefore, HILPDA is a polypeptide drug with significant medical and commercial value. Attached Figure Description

[0031] Figure 1 A diagram illustrating the absorption strategy of apoliposomes to assess whether HILPDA promotes the formation of APOE.

[0032] Figures 2A-2D This diagram illustrates the uptake of APOE by recipient cells in cell culture medium with and without HILPDA. In the diagram, 2A: 786-O; 2B: 769-P; 2C: HuH7; 2D: ES-2. Detailed Implementation

[0033] The following detailed embodiments are merely illustrative in nature and are not intended to limit the invention or its uses. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following detailed embodiments or examples.

[0034] Example

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] Example 1: Construction of a eukaryotic expression plasmid for the HILPDA-Flag fusion protein

[0038] 1) The DNA sequence corresponding to the human HILPDA-Flag fusion protein was directly synthesized and expressed in GENEWIZ.

[0039] ATGAAGCATGTGTTGAACCTCTACCTGTTAGGTGTGGTACTGACCCTACTCTCCATCTTCGTTAGAGTGATGGAGTCCCTAGAGGGCTTACTAGAGAGCCCATCGCCTGGGACCTCCTGGACCACCAGAAGCCAACTAGCCAACACAGAGCCCACCAAGGGCCTTCCAGACCATCCATCCAGAAGCATGGATTACAAGGATGACGACGATAAGTGA (Note: HILPDA; Flag; Stop codon) (SEQ ID NO: 12).

[0040] 2) Design the corresponding primers (forward: ATAGAAGATTCTAGA ATGAAGCATGTGTTGAACCTCTAC(SEQ IDNO:13); Reverse: CGCGGCCGCGGATCC The synthesized DNA sequence tcaCTTATCGTCGTCATCCTTGTAATC (SEQ ID NO:14) was used as a template for PCR amplification to obtain the target fragment. (The underlined portion indicates the homologous arm of the vector.)

[0041] 3) The eukaryotic expression vector pCDH-CMV-MCS-EF1-puro (catalog number: VT1480, UBO Biotechnology) was obtained by double digestion of the restriction endonucleases XbaI (Catalog No.: R0145L, NEB) and BamHI (Catalog No.: R3136L, NEB).

[0042] 4) The target fragment was ligated to the enzyme-digested linear vector using the homologous recombination method mediated by Exonuclease III (catalog number: 2170A, TAKARA), and the ligation product was transformed in E. coli DH5α. Positive colony clones were obtained on ampicillin-resistant LB solid medium plates.

[0043] 5) Amplify positive colony clones and extract plasmids using a plasmid purification kit.

[0044] 6) Sequencing verification to obtain the correct HILPDA fusion protein expression plasmid.

[0045] Example 2: Construction of GFP-Flag fusion protein expression plasmid

[0046] 1) The GFP gene sequence is derived from the vector pCDH-CMV-MCS-EF1-GFP+Puro (catalog number: VT8070, Youbao Biotechnology), and the specific sequence is as follows:

[0047] (Lowercase letters indicate stop codons) (SEQ ID NO:15).

[0048] 2) Design the corresponding primers (forward: ATAGAAGATTCTAGA ATGGTGAGCAAGGGCGAGGA(SEQ ID NO:16); Reverse: Using the GFP DNA sequence described above as a template, PCR amplification was performed to obtain the target fragment GFP-Flag sequence. (The underlined part indicates the vector homologous arm, and the bold part indicates the Flag sequence).

[0049] 3) The eukaryotic expression vector pCDH-CMV-MCS-EF1-puro (catalog number: VT1480, UBO Biotechnology) was obtained by double digestion of the restriction endonucleases XbaI (Catalog No.: R0145L, NEB) and BamHI (Catalog No.: R3136L, NEB).

[0050] 4) The target fragment was ligated to the enzyme-digested linear vector using the homologous recombination method mediated by Exonuclease III (catalog number: 2170A, TAKARA), and the ligation product was transformed in E. coli DH5α. Positive colony clones were obtained on ampicillin-resistant LB solid medium plates.

[0051] 5) Amplify positive colony clones and extract plasmids using a plasmid purification kit.

[0052] 6) Sequencing verification to obtain the correct GFP-Flag fusion protein expression plasmid.

[0053] Example 3: Construction of expression plasmids for fusion proteins corresponding to three different alleles of APOE2 / 3 / 4-3xHA

[0054] 1) The APOE3 gene sequence is derived from the vector pDONR223-APOE (catalog number: G118334, Youbao Biotechnology). The specific sequence is as follows:

[0055] ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGG TGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAGGAGTTGAAGGCCTACAAATCGGAACTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTG TGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAG CGC CTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGG AGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCACtga (lowercase letters are stop codons, and the underlined parts represent the different alleles of APOE. In this sequence, it specifically refers to APOE3) (SEQ ID NO:18).

[0056] 2) Design the corresponding APOE3 primers (forward: ATAGAAGATTCTAGA ATGAAGGTTCTGTGGGCTGCG(SEQID NO:19), reverse: CTTCCATGGCTCGAG The APOE3 DNA sequence (SEQ ID NO: 20) was used as a template for PCR amplification to obtain the target fragment APOE3 sequence. (The underlined part indicates the vector homologous arm).

[0057] 3) The gene sequences of APOE2 and APOE4 are obtained by point mutation of the APOE3 gene sequence.

[0058] (1) Design point mutation primers

[0059] APOE2, forward: ATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGC (SEQ ID NO: 21),

[0060] Reverse: GCCTGGTACACTGCCAGGCACTTCTGCAGGTCATCGGCAT (SEQ ID NO: 22);

[0061] APOE4, forward: GGCCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTA (SEQ ID NO: 23),

[0062] Reverse: TACTGCACCAGGCGGCCGCGCACGTCCTCCATGTCCGCGC(SEQ ID NO:24)

[0063] (2) Fragment 1 was amplified by PCR using the APOE3 forward primer + APOE2 reverse primer with the APOE3 gene sequence as a template; simultaneously, fragment 2 was amplified by PCR using the APOE2 forward primer + APOE3 reverse primer with the APOE3 gene sequence as a template. Finally, PCR amplification was performed using the mixture of fragment 1 and fragment 2 as a template with the APOE3 forward primer + APOE3 reverse primer to obtain the sequence of the target fragment APOE2. The specific sequence of APOE2 is as follows:

[0064] ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGG TGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAGGAGTTGAAGGCCTACAAATCGGAACTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTG TGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAG TGC CTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGG AGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCACtga (lowercase letters are stop codons, and the underlined parts represent the different alleles of APOE. In this sequence, it specifically refers to APOE2) (SEQ ID NO:25).

[0065] (3) Fragment 1 was amplified by PCR using the APOE3 forward primer + APOE4 reverse primer with the APOE3 gene sequence as a template; simultaneously, fragment 2 was amplified by PCR using the APOE4 forward primer + APOE3 reverse primer with the APOE3 gene sequence as a template. Finally, PCR amplification was performed using the mixture of fragment 1 and fragment 2 as a template with the APOE3 forward primer + APOE3 reverse primer to obtain the sequence of the target fragment APOE4.

[0066] The specific sequence of APOE4 is as follows:

[0067] ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAGGAGTTGAAGGCCTACAAATCGGAACTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTG CGC GGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAG CGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGG AGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCACtga (lowercase letters are stop codons, and the underlined parts represent the different alleles of APOE. In this sequence, it specifically refers to APOE4) (SEQ ID NO:26).

[0068] 4) The eukaryotic expression vector pCDH-CMV-MCS-EF1-3xHA-puro (which was self-constructed on the above pCDH-CMV-MCS-EF1-puro vector) was obtained by double digestion with restriction endonucleases XbaI (Catalog No.: R0145L, NEB) and XhoI (Catalog No.: R0146L, NEB). The vector pCDH-CMV-MCS-EF1-3xHA-puro was constructed as follows: First, the vector pCDH-CMV-MCS-EF1-puro was double-digested with XhoI (catalog number: R0145L, NEB) and NotI (catalog number: R3189L, NEB) to obtain a linear vector. Then, the 3xHA sequence (TACCCTTATGACGTACCTGACTATGCTGGCGCCTACCCTTATGACGTACCTGACTATGCTGGATACCCTTATGACGTACCTGACTATGCTtaa (SEQ ID NO:27)) was ligated to the linear vector pCDH-CMV-MCS-EF1-puro to form a vector with a 3xHA tag at the carboxyl terminus, namely pCDH-CMV-MCS-EF1-3xHA-puro.

[0069] 5) The target fragments APOE2 / APOE3 / APOE4 were ligated with the enzyme-digested linear vector pCDH-CMV-MCS-EF1-3xHA-puro using the homologous recombination method mediated by Exonuclease III (catalog number: 2170A, TAKARA). The ligation product was transformed in E. coli DH5α and positive colony clones were obtained on ampicillin-resistant LB solid medium plates.

[0070] 6) Amplify positive colony clones and extract plasmids using a plasmid purification kit.

[0071] 7) Sequencing verification to obtain the correct APOE2-3xHA / APOE3-3xHA / APOE4-3xHA fusion protein expression plasmid.

[0072] Example 4: Secretory HILPDA promotes the clearance of apolipoproteins and apoliposomes in serum.

[0073] 1) HEK293T cells were cultured in DMEM medium in 10cm cell culture dishes.

[0074] 2) In the above cells, co-transfect plasmids expressing GFP-Flag, HILPDA-Flag, and three different alleles of APOE2 / 3 / 4-3xHA into the cells respectively.

[0075] 3) After 12 hours, replace the old culture medium in 1) with fresh DMEM cell culture medium.

[0076] 4) After culturing for another 48 hours, collect the culture medium from cells cultured in step 3) above, and use this medium to directly culture recipient cells that have been pre-digested with trypsin containing 0.25% EDTA, such as 786-O (catalog number: THu186, Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), 769-P (catalog number: THu215, Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), HuH7 (catalog number: THu182, Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and ES-2 (catalog number: TCHu111 Wild-type cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences).

[0077] 5) After 24 hours of culture, immunofluorescence staining experiments were performed using anti-Flag (catalog number: F1804, Sigma; used to detect GFP-Flag or HILPDA-Flag fusion proteins) and anti-HA antibody (catalog number: ab9110, abcam; used to detect fusion proteins expressed by three different alleles of APOE2 / 3 / 4-3xHA).

[0078] 6) Observe the positioning of APOE-3xHA and HILPDA-Flag using laser confocal microscopy (e.g., Figures 2A-2D (As shown).

[0079] Figure 1 This study demonstrates an absorption strategy for assessing whether HILPDA promotes the formation of apoliposomes from APOE. Figures 2A-2D This shows the uptake of APOE by recipient cells in cell culture medium with and without HILPDA.

[0080] Experimental results showed that, compared with recipient cells such as 786-O, 769-P, Huh7, and ES-2 cultured in the medium for HEK293T cells co-expressing GFP-Flag and APOE2 / 3 / 4-3xHA proteins, recipient cells cultured in the medium for HEK293T cells co-expressing HILPDA-Flag and APOE2 / 3 / 4-3xHA proteins exhibited more and stronger fluorescence signals. This indicates that in the presence of HILPDA, recipient cells absorbed more apolipoproteins APOE2 / 3 / 4 (illustrated in the anti-HA antibody group), and also suggests that HILPDA promotes the clearance of apolipoproteins and the apoliposomes they form in the culture medium or serum.

[0081] Without being limited to any particular theory, the inventors hypothesize that the mechanism of action of secretory HILPDA is to promote the absorption of lipid particles from the blood by human cells through a series of extracellular and intracellular signal transduction pathways, thereby playing a preventive or therapeutic role in hyperlipidemia, atherosclerosis, and related complications. Since this naturally occurring small protein or polypeptide originates from the human body, it should exhibit low toxicity after being administered to patients, making it suitable for clinical translation and widespread application. Therefore, HILPDA is a polypeptide drug with significant medical and commercial value. SEQUENCE LISTING <110> Westlake University <120> Application of hypoxia-induced lipid droplet-associated protein (HILPDA) in the prevention or treatment of hyperlipidemia-related diseases <130> DI21-0616-XC37 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> PRT <213> Human (Homo sapiens) <400> 1 Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln Leu Ala Asn 35 40 45 Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser Arg Ser Met 50 55 60 <210> 2 <211> 64 <212> PRT <213> Mus musculus <400> 2 Met Lys Phe Met Leu Asn Leu Tyr Val Leu Gly Ile Met Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Gly Gly Leu Leu Glu 20 25 30 Ser Pro Leu Pro Gly Ser Ser Trp Ile Thr Arg Gly Gln Leu Ala Asn 35 40 45 Thr Gln Pro Pro Lys Gly Leu Pro Asp His Pro Ser Arg Gly Val Gln 50 55 60 <210> 3 <211> 63 <212> PRT <213> Green monkey (Chlorocebus sabaeus) <400> 3 Met Lys His Met Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Asn Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln Leu Ala Asn 35 40 45 Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser Arg Ser Ile 50 55 60 <210> 4 <211> 63 <212> PRT <213> Rhinopithecus roxellana <400> 4 Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Asn Pro Leu Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln Leu Thr Asn 35 40 45 Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser Arg Ser Met 50 55 60 <210> 5 <211> 63 <212> PRT <213> Cynomolgus macaque (Macaca fascicularis) <400> 5 Met Lys His Met Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Asn Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln Leu Ala Asn 35 40 45 Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser Arg Ser Met 50 55 60 <210> 6 <211> 62 <212> PRT <213> Domestic pig (Sus scrofa) <400> 6 Met Lys Gln Val Leu Gln Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Val Phe Val Arg Leu Met Glu Thr Leu Gly Gly Leu Leu Glu 20 25 30 Ser Pro Ser Pro Gly Ser Phe Trp Thr Thr Arg Gly Gln Leu Ala Asn 35 40 45 Thr Glu Ala Lys Ser Leu Pro Glu His Pro Ser Arg Gly Val 50 55 60 <210> 7 <211> 71 <212> PRT <213> Dog (Canis familiaris) <400> 7 Met Lys Pro Met Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Leu Met Glu Ser Leu Gly Gly Ile Leu Glu 20 25 30 Ser Pro Leu Leu Gly Ser Ser Trp Thr Thr Arg Gly His Thr Ala Ser 35 40 45 Ala Glu Pro Pro Arg Ala Phe Gln Thr Ile His Pro Glu Arg Cys Asp 50 55 60 Lys Thr Ser Leu His Gln Leu 65 70 <210> 8 <211> 71 <212> PRT <213> Goat (Capra hircus) <400> 8 Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Gly Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Leu Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Ser Pro Ser Pro Gly Ser Ser Trp Ala Thr Arg Gly Gln Leu Ala Asn 35 40 45 Thr Glu Pro Pro Arg Ala Phe Gln Thr Ile His Pro Glu Gly Cys Asp 50 55 60 Lys Thr Ser Leu His Arg Leu 65 70 <210> 9 <211> 71 <212> PRT <213> Horse (Equus caballus) <400> 9 Met Lys His Ile Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Leu Met Glu Ser Leu Glu Gly Leu Leu Glu 20 25 30 Ser Pro Ser Pro Gly Ser Ser Trp Thr Thr Arg Ser Gln Leu Val Ser 35 40 45 Thr Arg Pro Pro Arg Ala Phe Gln Thr Ile His Pro Glu Gly Cys Asp 50 55 60 Lys Thr Ser Val His Arg Leu 65 70 <210> 10 <211> 98 <212> PRT <213> Ox (Bos taurus) <400> 10 Met Lys His Met Leu Asn Leu Tyr Leu Leu Gly Gly Val Met Thr Leu 1 5 10 15 Leu Ser Ile Phe Ile Gly Leu Met Glu Ser Leu Glu Ala Cys Trp Arg 20 25 30 Ala Cys Leu Leu Gly Ala Pro Gly Pro Pro Glu Val Ser Leu Pro Thr 35 40 45 Gln Ser Pro Gln Gly Ser Ser Arg Pro Ser Ile Gln Arg Gly Val Ile 50 55 60 Arg Pro Pro Ser Thr Val Tyr Asn Leu Gly Thr Val Ser Lys Ile Pro 65 70 75 80 Pro Thr Leu Pro Val Pro Gly Gln Ala Ser Ser Thr Ser Val Leu Ser 85 90 95 Glu Asn <210> 11 <211> 64 <212> PRT <213> Oryctolagus cuniculus <400> 11 Met Lys His Val Leu Asn Leu Tyr Val Leu Gly Val Val Leu Ala Leu 1 5 10 15 Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Ser Leu Val Glu 20 25 30 Ser Pro Ser Ala Gly Asn Ser Trp Ser Thr Arg Gly Gln Leu Ala Gly 35 40 45 Ala Glu Pro Pro Lys Gly Leu Pro Asp Pro Pro Ser Arg Gly Met Arg 50 55 60 <210> 12 <211> 216 <212> DNA <213> Artificial sequence <220> <223> DNA sequence corresponding to the expression of human HILPDA-Flag fusion protein <400> 12 atgaagcatg tgttgaacct ctacctgtta ggtgtggtac tgaccctact ctccatcttc 60 gttagagtga tggagtccct agagggctta ctagagagcc catcgcctgg gacctcctgg 120 accaccagaa gccaactagc caacacagag cccaccaagg gccttccaga ccatccatcc 180 agaagcatgg attacaagga tgacgacgat aagtga 216 <210> 13 <211> 39 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> 13 atagaagatt ctagaatgaa gcatgtgttg aacctctac 39 <210> 14 <211> 42 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> 14 cgcggccgcg gatcctcact tatcgtcgtc atccttgtaa tc 42 <210> 15 <211> 720 <212> DNA <213> Artificial sequence <220> <223> GFP gene sequence <400> 15 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 16 <211> 35 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> 16 atagaagatt ctagaatggt gagcaagggc gagga 35 <210> 17 <211> 62 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> 17 cgcggccgcg gatccttact tatcgtcgtc atccttgtaa tccttgtaca gctcgtccat 60 gc 62 <210> 18 <211> 954 <212> DNA <213> Artificial sequence <220> <223> APOE3 gene sequence <400> 18 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccgggagccc gaggctcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagcgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctga 954 <210> 19 <211> 36 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> 19 atagaagatt ctagaatgaa ggttctgtgg gctgcg 36 <210> 20 <211> 35 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> 20 cttccatggc tcgaggtgat tgtcgctggg cacag 35 <210> twenty one <211> 40 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> twenty one atgccgatga cctgcagaag tgcctggcag tgtaccaggc 40 <210> twenty two <211> 40 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> twenty two gcctggtaca ctgccaggca cttctgcagg tcatcggcat 40 <210> twenty three <211> 40 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> twenty three gcgcggacat ggaggacgtg cgcggccgcc tggtgcagta 40 <210> twenty four <211> 40 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> twenty four tactgcacca ggcggccgcg cacgtcctcc atgtccgcgc 40 <210> 25 <211> 954 <212> DNA <213> Artificial sequence <220> <223> APOE2 sequence <400> 25 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctga 954 <210> 26 <211> 954 <212> DNA <213> Artificial sequence <220> <223> Sequence of APOE4 <400> 26 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa ggagttgaag gcctacaaat cggaactgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgcgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagcgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ccccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 [[ID=...]] gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctga 954 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctga 954 <210> 27<210> 27 <211> 93<211> 93 <212> DNA<212> DNA <213> Artificial Sequence<213> Artificial Sequence <220> <220> <223> 3xHA Sequence <223> 3xHA Sequence <400> 27 <400> 27 tacccttatg acgtacctga ctatgctggc gcctaccctt atgacgtacc tgactatgct 60 tacccttatg acgtacctga ctatgctggc gcctaccctt atgacgtacc tgactatgct 60 ggataccctt atgacgtacc tgactatgct taa 93 ggataccctt atgacgtacc tgactatgct taa 93

Claims

1. Use of hypoxia-inducible lipid droplet-associated protein in the preparation of a medicament for preventing or treating hyperlipidemia, wherein, The amino acid sequence of the hypoxia-inducible lipid droplet-associated protein is shown as SEQ ID NO:

1.

2. Use according to claim 1, wherein, The hypoxia-inducible lipid droplet-associated protein is further modified.

3. Use according to claim 1 or 2, wherein, The drug is for human.

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