Truncated mutants of chicken hepatocyte growth factor and use thereof in the prevention and treatment of liver-related diseases

Abstract

The application relates to the technical field of biological medicine, and particularly provides a truncated mutant of chicken hepatocyte growth factor and application thereof in preventing and treating liver-related diseases. A nucleic acid molecule constructed based on the truncated mutant of the chicken hepatocyte growth factor and taking E. coli as an expression vector not only realizes efficient expression of the truncated mutant of the chicken hepatocyte growth factor, lays a foundation for industrial application of the truncated mutant of the chicken hepatocyte growth factor, but also has significant biological activity.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically providing a truncated mutant of chicken liver cell growth factor and its use in the prevention and treatment of liver-related diseases. Background Technology

[0002] Hepatocyte growth factor (HGF) is a glycosylated protein with a complex structure, consisting of a heterodimer composed of a 60 kDa α chain and a 30 kDa β chain. The α chain contains an N-terminal hairpin structure and four kringle domains, while the β chain contains a serine protease-like domain.

[0003] HGF is mainly secreted by hepatic interstitial cells and plays an important role in cell proliferation, angiogenesis, liver regeneration, anti-inflammation and anti-fibrosis, as well as cellular immune regulation.

[0004] Currently, the production of HGF mainly includes: (1) separation and extraction from liver or blood components, but this is costly, complex, and prone to introducing foreign pathogens; (2) obtaining recombinant HGF protein using gene recombination technology. Since HGF is a large molecule protein and involves complex post-translational modifications, its production is usually limited to insect cells or mammalian cells, which makes the production process of HGF complex and costly, thus seriously affecting its widespread application. Summary of the Invention

[0005] In view of this, this application provides a truncated mutant of chicken hepatocyte growth factor and its use in the prevention and treatment of liver-related diseases. The nucleic acid molecule constructed based on the truncated mutant of chicken hepatocyte growth factor of this application, using *E. coli* as an expression vector, not only achieves efficient expression of the truncated mutant of chicken hepatocyte growth factor, laying the foundation for its industrial application, but also demonstrates that the truncated mutant of chicken hepatocyte growth factor exhibits significant biological activity.

[0006] In a first aspect, this application provides a truncated mutant of chicken hepatocyte growth factor, the amino acid sequence of which is shown in SEQ ID NO.1; the truncated mutant is selected from the amino acid sequence from position 28 to position 204 in SEQ ID NO.1 in the direction from N-terminus to C-terminus, and the point mutations in the truncated mutant include: leucine at position 100 being mutated to methionine and / or threonine at position 101 being mutated to serine, lysine at position 132 being mutated to glutamic acid, and alanine at position 134 being mutated to glutamic acid.

[0007] In some alternative embodiments, the truncated mutant is selected from the amino acid sequence from position 28 to position 204 in SEQ ID NO.1, in the direction from N-terminus to C-terminus, and the point mutations in the truncated mutant include: leucine at position 100 being mutated to methionine, lysine at position 132 being mutated to glutamic acid, and alanine at position 134 being mutated to glutamic acid.

[0008] In some specific embodiments, the truncated mutant is selected from the amino acid sequence from position 28 to position 204 of SEQ ID NO. 1, from N-terminus to C-terminus, and the point mutations in the truncated mutant include: threonine at position 101 mutated to serine, lysine at position 132 mutated to glutamic acid, and alanine at position 134 mutated to glutamic acid. In some optional embodiments, the truncated mutant is selected from the amino acid sequence from position 28 to position 204 of SEQ ID NO. 1, from N-terminus to C-terminus, and the point mutations in the truncated mutant include: leucine at position 100 mutated to methionine, threonine at position 101 mutated to serine, lysine at position 132 mutated to glutamic acid, and alanine at position 134 mutated to glutamic acid.

[0009] In some alternative embodiments, the truncated mutant is selected from any of the amino acid sequences shown in SEQ ID NO. 6 to 8.

[0010] In some specific embodiments, the truncated mutant is selected from the amino acid sequence shown in SEQ ID NO. 6.

[0011] In some specific embodiments, the truncated mutant is selected from the amino acid sequence shown in SEQ ID NO.7.

[0012] In some specific embodiments, the truncated mutant is selected from the amino acid sequence shown in SEQ ID NO.8.

[0013] Secondly, this application provides a nucleic acid molecule encoding the truncated mutant described in the first aspect.

[0014] In some alternative embodiments, the nucleic acid molecule is selected from the nucleotide sequences shown in any of SEQ ID NO. 12 to 14.

[0015] In some specific embodiments, the nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.12.

[0016] In some specific embodiments, the nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.13.

[0017] In some specific embodiments, the nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.14.

[0018] Thirdly, this application provides a recombinant plasmid comprising the nucleic acid molecules described in the second aspect.

[0019] Fourthly, this application provides a gene expression vector, characterized in that the gene expression vector comprises the nucleic acid molecule described in the second aspect.

[0020] Fifthly, this application provides the use of the truncated mutant described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the recombinant plasmid described in the third aspect, and / or the gene expression vector described in the fourth aspect in the preparation of products for the prevention and treatment of liver-related diseases.

[0021] In some alternative embodiments, the liver-related diseases include liver injury, liver fibrosis, chronic liver failure, and acute liver failure. In some specific embodiments, the liver-related diseases are selected from liver injury.

[0022] In a sixth aspect, this application provides a vaccine composition comprising the truncated mutant described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the recombinant plasmid described in the third aspect, and / or the gene expression vector described in the fourth aspect.

[0023] In a seventh aspect, this application provides a pharmaceutical composition comprising the truncated mutant described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the recombinant plasmid described in the third aspect, and / or the gene expression vector described in the fourth aspect.

[0024] This application has the following beneficial effects:

[0025] First, the nucleic acid molecule constructed based on the truncated mutant of chicken hepatocyte growth factor in this application, using Escherichia coli as a gene expression vector, not only achieves efficient expression of the truncated mutant of chicken hepatocyte growth factor, laying the foundation for the industrial application of the truncated mutant of chicken hepatocyte growth factor, but also the truncated mutant of chicken hepatocyte growth factor has significant biological activity.

[0026] Second, this application uses a combination of "threonine at position 101 mutated to serine" and "lysine at position 132 mutated to glutamic acid, and alanine at position 134 mutated to glutamic acid" in the truncated mutant, which can significantly improve the treatment effect of liver injury. Attached Figure Description

[0027] Figure 1 The signal peptide prediction results are from the online tool SignalP-5.0 in Example 1.

[0028] Figure 2 These are the screening results for potentially beneficial mutation sites from Example 1.

[0029] Figure 3 This is the electrophoresis result of SDS-PAGE in Example 2. Detailed Implementation

[0030] This application discloses a truncated mutant of chicken hepatocyte growth factor and its use in the prevention and treatment of liver-related diseases. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The methods and applications of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this application.

[0031] To make the objectives, technical solutions, and advantages of this application clearer, the implementation schemes of this application will be further described in detail below with reference to the embodiments.

[0032] Example 1: Truncated form and truncated mutant of chicken liver cell growth factor:

[0033] The full-length amino acid sequence of chicken hepatocyte growth factor is shown in SEQ ID No. 1, which is derived from NM_001030370.5 (NCBI). From the N-terminus to the C-terminus, chicken hepatocyte growth factor contains an N-terminal hairpin structure and four Kringle domains.

[0034] The amino acid sequence containing the N-terminal hairpin and the first Kringle domain of chicken hepatocyte growth factor is shown in SEQ ID No. 2; based on the amino acid sequence SEQ ID No. 2, signal peptide prediction was performed using the online tool SignalP–5.0. Figure 1 The truncated form of chicken liver cell growth factor was obtained, and the amino acid sequence of the truncated form is shown in SEQ ID No. 3.

[0035] Based on the amino acid sequence SEQ ID No. 3, regions with strong signals were first screened using the online tool Predict Protein; then, based on these sites in high-signal regions, the potential beneficial or detrimental effects of amino acid substitutions in the truncated protein on protein structure and function were predicted using the online tools PROVEAN and PolyPhen-2, and potentially beneficial mutation sites were screened. Figure 2 (Table 1).

[0036] Table 1. Screening Results:

[0037]

[0038] Based on the mutation of lysine K at position 132 to glutamic acid E and alanine A at position 134 to glutamic acid E in chicken hepatocyte growth factor from N-terminus to C-terminus, five truncated mutants of chicken hepatocyte growth factor were constructed. The amino acid sequences of the five truncated mutants are shown in SEQ ID No. 4 to 8, respectively.

[0039] Table 2. Sequence information of amino acid sequences:

[0040]

[0041] The nucleotide sequence of the nucleic acid molecule encoding the truncated form SEQ ID NO.3 is shown in SEQ ID NO.9;

[0042] The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.4 is shown in SEQ ID NO.10;

[0043] The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO. 5 is shown in SEQ ID NO. 11;

[0044] The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO. 6 is shown in SEQ ID NO. 12;

[0045] The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.7 is shown in SEQ ID NO.13;

[0046] The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO. 8 is shown in SEQ ID NO. 14.

[0047] Table 3. Sequence information of nucleotide sequences:

[0048]

[0049] Unless otherwise stated, all nucleic acid molecules involved in this application were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Example 2: Construction of recombinant plasmids and gene expression vectors, and expression of target proteins:

[0051] Synthetically synthesized nucleic acid molecules and pET-28a plasmid were ligated using a double-digestion enzyme vector construction technique to construct a recombinant plasmid. Specifically, the synthesized nucleic acid molecules and pET-28a plasmid were double-digested with EcoRI and HindIII. The digestion products were separated by agarose gel electrophoresis, and the target band and vector band were recovered from the gel. Then, they were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-nucleic acid molecule.

[0052] The recombinant plasmid pET-28a- nucleic acid molecule was transformed into BL21(DE3) competent cells and screened to obtain positive bacterial strains containing the nucleic acid molecule (i.e., gene expression vectors).

[0053] Positive bacterial strains containing the nucleic acid molecule were inoculated into LB liquid medium and cultured overnight at 37°C with shaking. Then, the overnight culture was inoculated into fresh LB liquid medium at a volume ratio of 1:100 and cultured at 37°C with shaking for approximately 2 hours until OD (dose retardation) was achieved. 600nm The concentration was adjusted to approximately 0.6. Then, an inducer (isopropyl-beta-D-thiogalactoside, CAS number 367-93-1) was added to a final concentration of 0.5 mM, and expression was induced overnight before fermentation was terminated. The bacterial cultures before and after induction were collected, quantified, concentrated, and 5×SDS-PAGE protein loading buffer was added until the final buffer concentration was 1×SDS. The mixture was then boiled in a water bath for 10 min. Protein expression was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0054] The electrophoresis results of SDS-PAGE are as follows: Figure 3 As shown, lane 1 represents the Mark protein; lane 2 represents the uninduced protein expression lane of mutant nucleic acid molecule SEQ ID NO.13; lane 4 represents the induced protein expression lane of mutant nucleic acid molecule SEQ ID NO.10; lane 5 represents the induced protein expression lane of mutant nucleic acid molecule SEQ ID NO.11; and lane 6 represents the induced protein expression lane of mutant nucleic acid molecule SEQ ID NO.13. Figure 3 It can be seen that the protein sizes induced by the mutant nucleic acid molecules SEQ ID NO. 10, 11 and 13 are all as expected.

[0055] Therefore, it can be seen that mutant nucleic acid molecules SEQ ID NO.10, 11 and 13 induce expression and obtain the target protein (i.e., mutant truncated versions).

[0056] Example 3: Evaluation of the therapeutic effect of a truncated mutant of chicken hepatocyte growth factor on liver injury:

[0057] Six- to eight-week-old BALB / c mice were randomly divided into five groups of ten each (half male and half female): a blank control group, a control group, and three experimental groups. Each group of mice was administered the drug via intraperitoneal injection.

[0058] In the control group, each mouse was given corn oil for 6 weeks, twice a week, with an injection volume of 2 ml / kg (based on mouse body weight).

[0059] Each mouse in both the control and experimental groups was first injected with a 25% (wt) CCl4 solution in corn oil for 3 weeks, twice a week, with an injection volume of 2 ml / kg (based on mouse body weight). In week 4, each mouse in the experimental group was injected with PBS buffer containing the truncated mutant, at a dose of 10 μg (based on the weight of the truncated mutant) per mouse, twice a week for 3 weeks; the control group was injected with the same volume of PBS buffer without the truncated mutant, twice a week for 3 weeks. The clinical status of the mice in each group was observed and recorded, and the liver organ coefficient and blood liver function indicators were measured 24 hours after the last injection. The results of the liver organ coefficient and blood liver function indicators are shown in Table 4.

[0060] The organ coefficient is the ratio of the weight of a specific organ to the total body weight of an experimental animal. Normally, this ratio remains relatively constant. After an animal is exposed to a toxin, the weight of the damaged organ changes, thus altering the organ coefficient. An increased organ coefficient indicates congestion, edema, or hyperplasia / hypertrophy of the organ; a decreased organ coefficient indicates organ atrophy and other degenerative changes.

[0061] Blood liver function indicators include aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Among them, AST and ALT are sensitive indicators of hepatocellular damage.

[0062] Table 4. Results of liver organ coefficient and blood liver function indicators in mice:

[0063]

[0064] As shown in Table 4, the truncated mutants SEQ ID NO. 4, 7, and 11 in experimental groups 1–3 all demonstrated therapeutic effects on liver injury, especially the truncated mutant SEQ ID NO. 7 in experimental group 3. This indicates that using the combination of "threonine at position 101 mutated to serine" and "lysine at position 132 mutated to glutamic acid, and alanine at position 134 mutated to glutamic acid" in the truncated mutants can significantly improve the therapeutic effect on liver injury.

[0065] The foregoing has provided a detailed description of a truncated mutant of chicken hepatocyte growth factor and its use in the prevention and treatment of liver-related diseases. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this application. It should be noted that those skilled in the art can make several improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A truncated mutant of chicken liver cell growth factor, characterized in that, The amino acid sequence of the truncated mutant is shown as SEQ ID NO.

7.

2. A nucleic acid molecule encoding the truncated mutant according to claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

13.

4. A recombinant plasmid, characterized by comprising the nucleotide sequence of SEQ ID NO:

1. The recombinant plasmid comprises the nucleic acid molecule according to any one of claims 2 to 3.

5. A gene expression vector, characterized by comprising The gene expression vector comprises the nucleic acid molecule according to any one of claims 2 to 3.

6. Use of the truncated mutant according to claim 1, and / or the nucleic acid molecule according to any one of claims 2 to 3, and / or the recombinant plasmid according to claim 4, and / or the gene expression vector according to claim 5 in the manufacture of a medicament for treating a liver-related disease; the liver-related disease is liver injury, chronic liver failure or acute liver failure.

7. Use of the truncated mutant according to claim 1, and / or the nucleic acid molecule according to any one of claims 2 to 3, and / or the recombinant plasmid according to claim 4, and / or the gene expression vector according to claim 5 in the manufacture of a medicament for treating a liver-related disease; the liver-related disease is liver fibrosis.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the truncated mutant according to claim 1, and / or the nucleic acid molecule according to any one of claims 2 to 3, and / or the recombinant plasmid according to claim 4, and / or the gene expression vector according to claim 5.

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