Truncated mutant of chicken hepatocyte growth factor and application of truncated mutant in prevention and treatment of liver-related diseases

Abstract

The invention relates to the technical field related to biological medicine, and particularly provides a truncated mutant of a chicken hepatocyte growth factor and application of the truncated mutant in the aspect of preventing and treating liver related diseases. According to the nucleic acid molecule constructed based on the truncated mutant of the chicken hepatocyte growth factor, escherichia coli serves as an expression vector, efficient expression of the truncated mutant of the chicken hepatocyte growth factor is achieved, and a foundation is laid for industrial application of the truncated mutant of the chicken hepatocyte growth factor; and the truncated mutant of the chicken hepatocyte growth factor has remarkable biological activity.

Description

Technical Field

[0001] The present application relates to the technical field of biomedicine, and specifically provides a truncated mutant of chicken hepatocyte growth factor and its use in preventing and treating liver-related diseases. Background Art

[0002] Hepatocyte growth factor (HGF) is a glycosylated protein with a complex structure, consisting 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, and the β chain contains a serine protease-like domain.

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

[0004] Currently, the production of HGF mainly includes: (1) separation and extraction from liver or blood components, but the cost is high, the operation is complicated and it is easy to introduce foreign pathogens; (2) the use of genetic recombination technology to obtain recombinant HGF protein. Since HGF is a large molecular 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 complicated and costly, thus seriously affecting its widespread application. Summary of the invention

[0005] In view of this, the present application provides a truncated mutant of chicken hepatocyte growth factor and its use in preventing and treating liver-related diseases. Based on the nucleic acid molecule constructed by the truncated mutant of chicken hepatocyte growth factor of the present application, Escherichia coli is used as an expression vector, which not only realizes the efficient expression of the truncated mutant of chicken hepatocyte growth factor, laying a 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.

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

[0007] In some optional embodiments, the truncated mutant is selected from the amino acid sequence at positions 28 to 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 mutates to methionine, lysine at position 132 mutates to glutamic acid, and alanine at position 134 mutates to glutamic acid.

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

[0009] In some optional embodiments, the truncation mutant is selected from the amino acid sequence shown in any one of SEQ ID NOs. 6 to 8.

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

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

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

[0013] In a second aspect, the present application provides a nucleic acid molecule encoding the truncated mutant described in the first aspect.

[0014] In some optional embodiments, the nucleic acid molecule is selected from the nucleotide sequence shown in any one of SEQ ID NOs. 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] In a third aspect, the present application provides a recombinant plasmid, wherein the recombinant plasmid comprises the nucleic acid molecule described in the second aspect.

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

[0020] In the fifth aspect, the present application provides a 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 preventing and treating liver-related diseases.

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

[0022] In the sixth aspect, the present application provides a vaccine composition, which includes 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 the seventh aspect, the present application provides a pharmaceutical composition, which includes 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: First, based on the nucleic acid molecule constructed from the truncated mutant of chicken hepatocyte growth factor of the present application, Escherichia coli is used as a gene expression vector, which 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.

[0025] Second, the present application uses a combination of "mutation of threonine at position 101 to serine" and "mutation of lysine at position 132 to glutamate, and mutation of alanine at position 134 to glutamate" in truncated mutants, which can significantly improve the therapeutic effect of liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the signal peptide prediction result of the online tool SignalP-5.0 in Example 1.

[0027] Figure 2 This is the screening result of the potentially beneficial mutation sites in Example 1.

[0028] Figure 3 This is the electrophoresis result of SDS-PAGE in Example 2. DETAILED DESCRIPTION

[0029] The present application discloses a truncated mutant of chicken hepatocyte growth factor and its use in preventing and treating liver-related diseases. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel 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 implement and apply the technology of this application.

[0030] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0031] Example 1: Truncated and truncated mutants of chicken hepatocyte growth factor: 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, the chicken hepatocyte growth factor comprises an N-terminal hairpin structure and four Kringle domains.

[0032] 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, the signal peptide was predicted using the online tool SignalP-5.0 ( Figure 1 ), and obtained a truncated form of chicken hepatocyte growth factor, the amino acid sequence of which is shown in SEQ ID No.3.

[0033] Based on the amino acid sequence SEQ ID No.3, we first used the online tool Predict Protein to screen out regions with strong signals; then based on the sites in these high-scoring regions, we used the online tools PROVEAN and PolyPhen-2 to predict the possible beneficial and harmful effects of amino acid substitutions in truncated forms on protein structure and function, and screened out potentially beneficial mutation sites ( Figure 2 , Table 1).

[0034] Table 1, screening results:

[0035] Based on the mutation of lysine K at position 132 to glutamate E and alanine A at position 134 to glutamate 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.

[0036] Table 2. Sequence information of amino acid sequence:

[0037] Wherein, the nucleotide sequence of the nucleic acid molecule encoding the truncated SEQ ID NO.3 is shown in SEQ ID NO.9; The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.4 is shown in SEQ ID NO.10; The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.5 is shown in SEQ ID NO.11; The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.6 is shown in SEQ ID NO.12; The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.7 is shown in SEQ ID NO.13; The nucleotide sequence of the nucleic acid molecule encoding the truncated mutant SEQ ID NO.8 is shown in SEQ ID NO.14.

[0038] Table 3. Sequence information of nucleotide sequences:

[0039] Unless otherwise specified, the nucleic acid molecules involved in this application were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0040] Example 2: Construction of recombinant plasmid and gene expression vector and expression of target protein: The artificially synthesized nucleic acid molecule and the pET-28a plasmid were connected using the double-cut enzyme vector construction technology to construct a recombinant plasmid. Specifically, the synthesized nucleic acid molecule and the pET-28a plasmid were double-digested with EcoRI and HindIII, the digested products were separated by agarose gel electrophoresis, the gel was cut to recover the target band and the vector band; and then connected with T4 DNA ligase to obtain the recombinant plasmid pET-28a-nucleic acid molecule.

[0041] The recombinant plasmid pET-28a-nucleic acid molecule was transformed into BL21 (DE3) competent cells, and screened and identified to obtain positive strains containing the nucleic acid molecule (i.e., the gene expression vector).

[0042] The positive bacteria containing the nucleic acid molecule were inoculated into LB liquid culture medium and cultured overnight at 37°C with shaking; then, the bacterial solution cultured overnight with shaking was inoculated into new LB liquid culture medium at a volume ratio of 1:100 and cultured at 37°C with shaking for about 2 hours until the OD 600nm The value was about 0.6; then, the inducer (isopropyl-beta-D-thiogalactoside, CAS No. 367-93-1) was added to a final concentration of 0.5mM, and the expression was induced overnight and then the fermentation was terminated. The bacterial solution before and after induced expression was taken, and after quantification, the bacterial solution was concentrated and added with 5×SDS-PAGE protein loading buffer until the final concentration of the buffer was 1×SDS, and it was placed in a boiling water bath and boiled for 10 minutes. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sodium dodecyl sulfate - polyacrylamide gel electrophoresis, SDS-PAGE) was used to detect protein expression.

[0043] Among them, the electrophoresis results of SDS-PAGE are as follows Figure 3 As shown, 1 represents the Mark protein lane; 2 represents the protein lane of the mutant nucleic acid molecule SEQ ID NO.13 that is not induced to express; 4 represents the protein lane of the mutant nucleic acid molecule SEQ ID NO.10 that is induced to express; 5 represents the protein lane of the mutant nucleic acid molecule SEQ ID NO.11 that is induced to express; and 6 represents the protein lane of the mutant nucleic acid molecule SEQ ID NO.13 that is induced to express. Figure 3 It can be seen that the sizes of proteins induced to express by the mutant nucleic acid molecules SEQ ID NOs. 10, 11 and 13 are all in line with expectations.

[0044] It can be seen that the mutant nucleic acid molecules SEQ ID NO. 10, 11 and 13 are induced to express and obtain the target protein (ie, the mutant truncation).

[0045] Example 3: Evaluation of the therapeutic effect of truncated mutants of chicken hepatocyte growth factor on liver damage: BALB / c mice aged 6 to 8 weeks were randomly divided into 5 groups, 10 mice in each group (half male and half female), including 1 blank group, 1 control group and 3 experimental groups. Each group of mice was intraperitoneally injected with drugs.

[0046] Each mouse in the blank group was given corn oil twice a week for 6 weeks, with each injection volume of 2 ml / kg (based on mouse body weight).

[0047] Each mouse in the control group and the experimental group was first injected with a 25% (wt) CCl4 corn oil solution for 3 weeks, twice a week, and the injection volume was 2 ml / kg (based on the weight of the mouse). In the fourth week, each mouse in the experimental group was injected with a PBS buffer containing a truncated mutant, with a dose of 10 ug (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 a truncated mutant, twice a week, for 3 weeks. The clinical status of mice in each group was observed and recorded, and the liver organ coefficient and blood liver function index of the mice were detected 24 hours after the last injection. The test results of the liver organ coefficient and blood liver function index of the mice are shown in Table 4.

[0048] Among them, the organ coefficient is the ratio of the weight of a certain organ of the experimental animal to its body weight. Under normal circumstances, the ratio of each organ to body weight is relatively constant. After the animal is poisoned, the weight of the damaged organ changes, so the organ coefficient also changes accordingly. An increase in the organ coefficient indicates congestion, edema, or hyperplasia of the organ; a decrease in the organ coefficient indicates organ atrophy and other degenerative changes.

[0049] Blood liver function indicators include aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Among them, AST and ALT are sensitive indicators of liver cell damage.

[0050] Table 4. Test results of liver organ coefficient and blood liver function index of mice:

[0051] As can be seen from Table 4, in the treatment of liver injury, the truncated mutants SEQ ID NO. 4, 7 and 11 in experimental groups 1 to 3 all played a role in treating liver injury, especially the truncated mutant SEQ ID NO. 7 in experimental group 3. It can be seen that the combination of "mutation of threonine at position 101 to serine" and "mutation of lysine at position 132 to glutamic acid, and mutation of alanine at position 134 to glutamic acid" in the truncated mutants can significantly improve the therapeutic effect of liver injury.

[0052] The above is a detailed introduction to a truncated mutant of chicken hepatocyte growth factor provided by the present application and its use in preventing and treating liver-related diseases. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A truncated mutant of chicken hepatocyte growth factor, characterized in that: The amino acid sequence of the chicken hepatocyte growth factor is shown in SEQ ID NO.1; from the N-terminus to the C-terminus, the truncated mutant is selected from the amino acid sequence at positions 28 to 204 in SEQ ID NO.1, and the point mutations in the truncated mutant include: the leucine at position 100 mutates to methionine and / or the threonine at position 101 mutates to serine, the lysine at position 132 mutates to glutamic acid, and the alanine at position 134 mutates to glutamic acid.

2. The truncated mutant according to claim 1, characterized in that From the N-terminus to the C-terminus, the truncated mutant is selected from the amino acid sequence from position 28 to position 204 in SEQ ID NO.1, and the point mutations in the truncated mutant include: the threonine at position 101 mutates to serine, the lysine at position 132 mutates to glutamic acid, and the alanine at position 134 mutates to glutamic acid.

3. The truncated mutant according to claim 1, characterized in that The truncated mutant is selected from the amino acid sequence shown in any of SEQ ID NO. 6 to 8; Optionally, the truncation mutant is selected from the amino acid sequence shown in SEQ ID NO.

7.

4. A nucleic acid molecule encoding the truncation mutant according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule is selected from the nucleotide sequence shown in any one of SEQ ID NOs. 12 to 14; Optionally, the nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.

13.

6. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleic acid molecule according to any one of claims 4 to 5.

7. A gene expression vector, characterized in that: The gene expression vector comprises the nucleic acid molecule according to any one of claims 4 to 5.

8. Use of the truncated mutant according to any one of claims 1 to 3, and / or the nucleic acid molecule according to any one of claims 4 to 5, and / or the recombinant plasmid according to claim 6, and / or the gene expression vector according to claim 7 in the preparation of products for preventing and treating liver-related diseases; Optionally, the liver-related diseases include liver damage, liver fibrosis, chronic liver failure and acute liver failure.

9. A vaccine composition, characterized in that The vaccine composition comprises the truncated mutant as described in any one of claims 1 to 3, and / or the nucleic acid molecule as described in any one of claims 4 to 5, and / or the recombinant plasmid as described in claim 6, and / or the gene expression vector as described in claim 7.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the truncated mutant according to any one of claims 1 to 3, and / or the nucleic acid molecule according to any one of claims 4 to 5, and / or the recombinant plasmid according to claim 6, and / or the gene expression vector according to claim 7.

Images