A polypeptide Hn1 and its application in preparing drugs for preventing and treating liver damage

Abstract

The present invention discloses a polypeptide Hn1 and its application in preparing a drug for preventing and treating liver damage, wherein the amino acid sequence of the polypeptide Hn1 is shown in SEQ ID NO.1. The present invention constructs an expression vector of the Hn1 polypeptide, uses BL12 (DE3) Escherichia coli for prokaryotic expression, and purifies the Hn1 recombinant protein using Ni-NTA purification technology. By establishing a hepatocyte injury model, the protective effect of the Hn1 polypeptide on damaged hepatocytes is studied. The results of in vitro cytology and in vivo animal experiments show that the Hn1 polypeptide can significantly reduce the damage of chemical poisons to hepatocytes, reduce the serum ALT and AST levels of experimental mice with acute liver damage induced by CCl4, improve the survival rate of model mice, and has a protective effect on acute liver damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a polypeptide Hn1 and an application thereof in preparing a drug for preventing and treating liver damage. Background Art

[0002] Acute liver injury (ALI) refers to the process in which the liver suffers pathological damage in a short period of time due to multiple causes such as infection, drug overdose, and exposure to toxins. The liver is an important organ with multiple functions such as secretion, detoxification, excretion, synthesis, biotransformation, and immunity. Compared with other organs, the liver is more susceptible to damage from drugs, alcohol, viruses, and toxic metabolites. The liver has a strong compensatory reserve function and regenerative capacity, and mild acute liver damage can be repaired by itself through liver cell proliferation. However, severe liver damage may lead to acute liver failure, a sharp decline in liver function, metabolic dysfunction, and may even cause serious complications such as abnormal coagulation and hepatic encephalopathy, posing a threat to the patient's life.

[0003] At present, the treatment of acute liver injury mainly includes traditional treatment options such as basic medical treatment, liver transplantation, and artificial liver replacement. However, these treatments have certain limitations, including the lack of specific drugs, scarcity of liver transplantation resources, and difficulty in postoperative monitoring. Therefore, the treatment of acute liver injury still faces severe challenges. It is particularly important to find specific, safe, and effective drugs for the treatment of acute liver injury. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a polypeptide Hn1 and its application in preparing a drug for preventing and treating liver damage.

[0005] In order to achieve the above technical objectives, the technical solution implemented by the present invention is:

[0006] The first object of the present invention is to provide a polypeptide Hn1, the amino acid sequence of the polypeptide Hn1 is shown as SEQ ID NO.1.

[0007] The second object of the present invention is to provide a nucleotide encoding the polypeptide Hn1.

[0008] The third object of the present invention is to provide a recombinant expression vector carrying the nucleotide.

[0009] The fourth object of the present invention is to provide a recombinant cell containing the recombinant expression vector.

[0010] The fifth object of the present invention is to provide a recombinant protein containing the polypeptide Hn1.

[0011] The sixth objective of the present invention is to provide the use of the polypeptide Hn1, nucleotides, recombinant expression vectors, and recombinant proteins in the preparation of drugs for treating liver damage.

[0012] The liver injury is acute liver injury.

[0013] The in vitro cytological experiment provided by the present invention shows that the Hn1 polypeptide can significantly reduce the damage of H2O2 or CCl4 to liver cells, and the protective effect of Hn1 on damaged liver cells is positively correlated with the dose of the drug. In vivo studies on mice with acute liver injury show that Hn1 can significantly reduce the serum ALT and AST levels of mice with acute liver injury induced by CCl4, improve the survival rate of model mice, and has a protective effect on acute liver injury, and its protective effect is related to the drug dose.

[0014] The seventh object of the present invention is to provide a pharmaceutical composition containing the above-mentioned polypeptide Hn1, nucleotides, recombinant expression vector or recombinant protein.

[0015] Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier, which includes but is not limited to a filler, an excipient, a stabilizer, a diluent, a binder, a lubricant or a surfactant.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention constructs an expression vector of Hn1 polypeptide, uses BL12 (DE3) Escherichia coli for prokaryotic expression, and purifies Hn1 recombinant protein by Ni-NTA purification technology. The present invention establishes a liver cell injury model to study the protective effect of Hn1 polypeptide on damaged liver cells.

[0018] (2) The results of in vitro cytological and in vivo animal experiments of the present invention show that Hn1 polypeptide can significantly reduce the damage of chemical poisons to liver cells, reduce the serum ALT and AST levels of mice with acute liver injury induced by CCl4, and improve the survival rate of model mice, thus having a protective effect on acute liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1The induced expression and purification profile of the Hn1 polypeptide in Example 1, wherein a is a Coomassie brilliant blue staining profile of the induced expression and purification of Hn1 (1: before induction; 2: after induction; 3: ultrasonic supernatant after induction; 4: ultrasonic precipitation after induction; 5-8: 50 / 150 / 250 / 500 mM imidazole eluent); b is a Coomassie brilliant blue staining identification profile; and c is a Western Blot identification profile (rabbit anti-His monoclonal antibody).

[0021] Figure 2 The effect of Hn1 polypeptide on the proliferation ability of AML12 cells damaged by 0.4mM-H2O2 in Example 3, wherein a is a CCK8 experimental detection showing that Hn1 polypeptide can significantly improve the viability of AML12 cells damaged by H2O2; b-1 and b-2 are immunocytochemistry experimental detections showing that Hn1 polypeptide can significantly enhance the proliferation ability of AML12 cells damaged by H2O2.

[0022] Figure 3 The protective effect of Hn1 polypeptide on liver damage in mice in Example 3, wherein a is the difference in body weight of mice in each group on the 7th day of CCl4 modeling; b is the effect of Hn1 polypeptide on the survival rate of mice with liver damage; c and d are the ALT and AST levels in the serum of mice in each group at different times after the mice were injected with 20% CCl4; e is the effect of Hn1 polypeptide on liver pathological morphology of mice with acute liver failure induced by CCl4. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0024] The term "polypeptide" in the present invention refers to a molecule composed of amino acids as basic units. The molecular weight of a polypeptide is between that of a chemical small molecule drug and a biological agent: compared with biological agents (such as antibodies, therapeutic proteins, etc.), it has a similar mode of action, but the immunogenicity of a polypeptide is lower, and because it can be chemically synthesized, its production cost is also lower; compared with chemical small molecule drugs, a polypeptide has a larger molecular weight, can effectively inhibit protein-protein interactions, has higher selectivity and specificity, and has lower concentrations and side effects.

[0025] The present invention also provides nucleotides encoding the above polypeptides. The nucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded.

[0026] The nucleotides encoding the polypeptide of the present invention include: coding sequences encoding only the polypeptide; coding sequences of the polypeptide and various additional coding sequences; coding sequences of the polypeptide (and optional additional coding sequences) and non-coding sequences.

[0027] The term "nucleotide encoding a polypeptide" may include nucleotides encoding the polypeptide, and may also include nucleotides additionally encoding and / or non-coding sequences.

[0028] The full-length nucleotide sequence or fragments thereof of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0029] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0030] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0031] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; animal cells such as CHO, COS7, 293 cells, etc.

[0032] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.

[0033] The present invention also provides a pharmaceutical composition, which contains the above-mentioned polypeptide or its active fragment or its fusion fragment, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.

[0034] The pharmaceutical composition of the present invention contains a safe and effective amount of the above-mentioned polypeptide of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): fillers, excipients, stabilizers, diluents, adhesives, lubricants, surfactants or combinations thereof. The amount of active ingredient administered is a therapeutically effective amount. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0035] When using a pharmaceutical composition, a safe and effective amount of the drug is administered to an individual. The specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skills of a skilled physician.

[0036] Example 1. Hn1 polypeptide

[0037] The present invention discloses that the molecular weight of the Hn1 polypeptide is 20.49 kDa, and it contains 180 amino acids. The specific amino acid sequence is shown in Table 1.

[0038] Table 1: Amino acid sequence of Hn1 polypeptide

[0039]

[0040] Example 2. Preparation of Hn1 polypeptide

[0041] According to the Hn1 gene sequence and PET-24a(+) plasmid sequence, upstream and downstream primers were designed, and NdeI restriction site was added to the upstream primer and Hind III restriction site was added to the downstream primer to construct the PET-24a(+) / Hn1 prokaryotic stable expression vector. pCold II and Hn1 were double-digested with restriction endonucleases Nde I and Hind III, respectively, and the double-digested products were purified. The recovered Hn1 double-digested fragment was connected to the pCold II linearized vector in a ligation system at a molar ratio of 5:1 for recombinant plasmid, and high-fidelity polymerase was added, mixed and placed at 37°C for 20 minutes. The pCold II / Hn1 recombinant expression vector was obtained. The obtained pCold II / HPPCn recombinant expression vector was transformed into Escherichia coli DH5α competent cells for prokaryotic expression. The Hn1 recombinant protein was purified using Ni-NTA purification technology.

[0042] Table 2: Upstream and downstream primers of Hn1

[0043]

[0044] Figure 1 The results show that: (1) Hn1 polypeptide is a soluble expression protein. The protein content in the ultrasonic supernatant after induced expression is significantly higher than that in the ultrasonic precipitate after induced expression, that is, Hn1 polypeptide is a soluble expression protein. Figure 1 (2) The purity of Hn1 peptide was the highest when eluted with 500 mmol / L imidazole (see Figure 1 (3) After a large number of induced expression and Ni-NTA column purification, the collected solution was dialyzed with PBS. Coomassie brilliant blue staining showed that the purity of Hn1 polypeptide was more than 90% (see Figure 1b), and Western Blot results showed that Hn1 peptide can be recognized by 6x-His-tag (ab213204) antibody (see Figure 1 c). In summary, the Hn1 polypeptide was successfully purified after identification. The Hn1 polypeptide after PBS dialyzation was filtered and sterilized using a 0.22 μm filter, and the concentration was measured and aliquoted and frozen for subsequent use.

[0045] Example 3. Application of Hn1 polypeptide in the study of protecting liver damage

[0046] 3.1 Protective effect of Hn1 peptide on damaged hepatocytes

[0047] In this study, normal mouse hepatocytes (AML12 cell line) were selected as hepatocyte model cells, 0.4 mM-H2O2 was used as hepatocyte damaging agent, CCK-8 experiment and immunocytochemistry experiment were used to detect hepatocyte viability and the protective effect of Hn1 on damaged hepatocytes.

[0048] (1) CCK-8 experiment verifies the protective effect of Hn1 on hepatocyte damage

[0049] 1) Cell line and culture medium: Mouse normal liver cell AML12 cells were used as the model cells of this study. The culture medium contained 89% DMEM / F12 (1:1), 10% high-quality fetal bovine serum, 1% insulin-transferrin-selenium (ITS-G) liquid culture medium supplement, and dexamethasone 40 ng / mL.

[0050] 2) Cell injury and injury protection grouping and treatment:

[0051] AML12 cell injury experimental groups: control group, H2O2 injury group, Hn1 low-dose group, Hn1 medium-dose group, and Hn1 high-dose group.

[0052] Control group (Ctrl group): AML12 cells were plated in a 96-well plate at 1000 cells / well and cultured normally.

[0053] Injury group (H2O2 group): AML12 cells were plated in a 96-well plate at 1000 cells / well, cultured for 12 h, and then replaced with a culture medium containing 0.4 mM-H2O2 solution. After injury for 6 h, H2O2 was removed and cultured for another 24 h.

[0054] The Hn1 polypeptide protection group (H2O2+Hn1 (ng / ml)) was divided into low-dose (25ng / mL), medium-dose (50ng / mL) and high-dose protection groups (100ng / mL): Cell injury was the same as the injury group. After removing H2O2, different concentrations of Hn1 polypeptide solutions were added to the culture medium and cultured for another 24h.

[0055] 3) CCK-8 detection and cell viability calculation: Prepare a volume ratio of fresh culture medium to CCK-8 solution of 9:1, discard the 96-well plate cell culture medium, quickly replace it with CCK-8 detection solution (100 μL / well), continue to culture for 3 hours, place it on the microplate reader and select the oscillation mixing mode, and measure the absorbance value of each well at 450nm. Calculate the cell proliferation activity according to the formula to evaluate the protective effect of Hn1 on liver cell damage. Cell viability calculation: Cell proliferation ability = [(A 实验组 -A 空白对照组) / (A 对照组 -A 空白对照组 )]×100%.

[0056] (2) Immunocytochemistry experiments verified the protective effect of Hn1 on hepatocyte damage

[0057] 1) Cell culture was the same as above, and the drug intervention groups were 25 ng / mL and 50 ng / mL dose groups.

[0058] 2) Immunocytochemistry experiments:

[0059] ① Cell fixation: discard the cell culture medium, wash with PBS for 5 times, 3 min each time, add 4% paraformaldehyde to fix for 20 min, and then wash with PBS for 5 times, 3 min each time.

[0060] ② Cell perforation: Add 0.1% Triton working solution, incubate on ice for 30 minutes, wash with PBS 5 times, 3 minutes each time.

[0061] ③ Removal of endogenous enzymes: Add 3% H2O2 solution, incubate at room temperature for 20 minutes to inactivate endogenous enzymes, and wash with PBS 5 times, 3 minutes each time.

[0062] ④ Blocking: Prepare 10% BSA blocking solution, add it into a 24-well plate, block at room temperature for 20 minutes, gently remove excess liquid, and do not wash.

[0063] ⑤ Primary antibody incubation: Transfer the cell slides to a glass slide and circle the incubation range with a histochemical pen. Dilute rabbit anti-Ki67 (ab16667) monoclonal antibody with 10% BSA solution at a ratio of 1:100, incubate overnight at 4°C (12h), collect the primary antibody (can be used 2-3 times), and wash 3 times with PBS, 2min / time.

[0064] ⑥ Secondary antibody incubation: dilute goat anti-rabbit secondary antibody (ab7090) at 1:500 with 10% BSA solution, incubate at 37°C for 1 hour, and wash with PBS 3 times, 2 minutes each time.

[0065] ⑦DAB color development: Prepare according to the dosage and instructions of the Zhongshan Jinqiao DAB kit. Mix the fresh DAB mixture thoroughly and add it dropwise to the cells. Incubate at room temperature in the dark for 5-10 minutes until the cell nucleus turns obviously brown. Wash with distilled water to terminate the reaction.

[0066] ⑧Observation: Add sealing liquid to the slide, slowly turn the successfully stained slide upside down on the sealing liquid (make sure there are no bubbles) to seal the slide, observe the cell status of each group under an ordinary optical microscope and take pictures for preservation.

[0067] The experimental results are as follows Figure 2 As shown, the results showed that compared with the Ctrl group, the cell viability and cell number of AML12 cells induced by 0.4mM-H2O2 were significantly reduced (##P<0.01). Compared with the injury group, after 6h of H2O2 injury and 24h of adding different concentrations of Hn1 peptide, the cell viability and cell number of AML12 cells were significantly increased, and there was a certain concentration dependence (*P<0.05, **P<0.01) ( Figure 2 a, b and c).

[0068] This indicates that Hn1 has a significant protective effect on hepatocytes damaged by H2O2, and its protective effect is positively correlated with the dose of Hn1.

[0069] (3) In vivo animal experiments to detect the protective effect of Hn1 peptide on chemically damaged liver

[0070] The specific method is as follows:

[0071] 1) Experimental mice: Male BALB / c mice, 8-9 weeks old, 20-23g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were housed in cages of 5-6 mice per cage, and were allowed to eat and drink water on their own. Animal experiments were conducted after 5 days of adaptive feeding, and the experimental period was 7 days. The bedding and food used for the mice in this study were all from the original company of the mice.

[0072] 2) Animal grouping and drug administration:

[0073] According to the random allocation principle, 26 mice / group were divided into the following three groups: negative control group, liver injury group, and Hn1 polypeptide protection group.

[0074] Negative control group (oil group): intraperitoneal injection of 10mL / kg olive oil; liver injury group (CCl4 group / Model group): intraperitoneal injection of 20% CCl4 solution at a dose of 10mL / kg to establish a CCl4-induced animal liver injury model; Hn1 polypeptide protection group: Hn1 polypeptide solution was injected into the tail vein at a dose of 0.5mg / kg and 1mg / kg at 1h, 12h, 24h, 36h, 48h, and 60h after the establishment of the CCl4 liver injury animal model.

[0075] 3) Preparation of reagents:

[0076] 20% CCl4 solution was prepared according to the volume ratio of CCl4 to olive oil of 1:4 and was thoroughly shaken and mixed before injection;

[0077] A solution of Hn1 polypeptide with a final concentration of 200 μg / mL was prepared using PBS.

[0078] 4) Weight monitoring and observation of the general condition of mice: observe their mental state, activities, eating, etc. regularly every day;

[0079] 5) Blood sample collection:

[0080] In this experiment, blood was collected from each group of mice by taking blood from the inner canthus. The blood volume was 50 μL-80 μL / mouse. The blood was collected before CCl4 injection and 6h, 12h, 1d, 2d, 4d, and 7d after CCl4 injection. The collected blood was placed in a 1.5 mL centrifuge tube at 4°C for 1 hour, centrifuged at 3000 rpm / min and 4°C for 10 minutes in a low-temperature centrifuge. The serum was gently aspirated and transferred to an RNAfree-100 μL tube and stored in a -20°C refrigerator for later use.

[0081] 6) Serum biochemical indicators AST and ALT determination:

[0082] The AST and ALT detection kits of Nanjing Jiancheng Bioengineering Institute were used to measure the AST and ALT of serum collected from each group and at each time point.

[0083] 7) Liver sampling:

[0084] On the second day after CCl4 injection, three mice were randomly selected from each group for liver sampling. The liver status was observed and photographed after each sampling. Finally, the liver was fixed in 4% paraformaldehyde for HE staining.

[0085] The experimental results are as follows Figure 3 The results showed that compared with the CCl4 group, the weight of mice in the Hn1 peptide groups at different concentrations was significantly higher than that in the CCl4 group ( * P<0.05, **P<0.01) (Figure a) Hn1 polypeptide increased the survival rate of mice with acute liver failure. Compared with the CCl4 group, the survival rates of mice with acute liver failure in the 0.5mg / kg and 1mg / kg Hn1 polypeptide groups were significantly increased (compared with the CCl4 group, Log-rank P=0.0498, Log-rank P=0.0174). The survival rate of mice with acute liver failure in the CCl4 group was 18.75%, and the survival rates of mice with acute liver failure in the 0.5mg / kg and 1mg / kg Hn1 polypeptide groups were 43.75% and 56.25%, respectively (Figure b). Compared with the CCl4 group, the ALT level in the serum of the Hn1 polypeptide injection group at 12h, 1d and 2d was lower than that of the CCl4 group; the AST level in the serum of the Hn1 polypeptide group at 1d and 2d was lower than that of the CCl4 group (*P<0.05, **P<0.01) (Figures c and d). HE staining results showed that compared with the Oil group, the liver cell gaps in the CCl4-treated group were enlarged, the nuclei were lightly stained, and the liver cell cords were disordered. Compared with the CCl4 group, the damage to the liver tissue structure of mice in the CCl4+Hn1 polypeptide group was significantly alleviated (Figure e).

[0086] In summary, in vivo animal experiments show that Hn1 polypeptide has a protective effect on chemically damaged animal livers.

[0087] Example 4, a pharmaceutical composition

[0088] The invention contains the polypeptide Hn1, nucleotides, recombinant expression vector or recombinant protein as active ingredients.

[0089] The pharmaceutical composition further contains a pharmaceutically acceptable carrier, which includes but is not limited to a filler, an excipient, a stabilizer, a diluent, a binder, a lubricant or a surfactant.

[0090] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide Hn1, characterized in that The amino acid sequence of the polypeptide Hn1 is shown in SEQ ID NO.

1.

2. A nucleotide sequence encoding the polypeptide Hn1 according to claim 1.

3. A recombinant expression vector carrying the nucleotide sequence of claim 2.

4. A recombinant cell containing the recombinant expression vector according to claim 3.

5. A recombinant protein containing the polypeptide Hn1 according to claim 1.

6. Use of any one of the polypeptide Hn1 according to claim 1, the nucleotide according to claim 2, the expression vector according to claim 3, the recombinant expression vector according to claim 4, and the recombinant protein according to claim 5 in the preparation of a drug for preventing and treating liver damage.

7. The use according to claim 6, characterized in that: The liver injury is acute liver injury.

8. A pharmaceutical composition comprising any one of the polypeptide Hn1 according to claim 1, the nucleotide according to claim 2, the expression vector according to claim 3, the recombinant expression vector according to claim 4, and the recombinant protein according to claim 5.

9. The pharmaceutical composition according to claim 8, characterized in that It also contains a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 8, characterized in that The carrier includes a filler, an excipient, a stabilizer, a diluent, a binder, a lubricant or a surfactant.

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