A polypeptide for delaying telomere shortening and its application

Abstract

The present invention relates to a polypeptide for delaying telomere shortening and its application, and belongs to the field of biotechnology. The present invention provides a polypeptide for delaying telomere shortening, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1. Studies have shown that the polypeptide has high biological activity and can significantly slow down the cell aging process by delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase, and inhibiting the expression level of β-galactosidase. In addition, the polypeptide is derived from the fermentation process of natural dairy products and has high safety and low risk of side effects. In addition, the polypeptide has a small molecular weight and is easily absorbed by the human body, which can improve the bioavailability of the drug. Therefore, the polypeptide has great application prospects in delaying telomere shortening, anti-aging, and the treatment of diseases related to telomere loss or dysfunction.

Description

Technical Field

[0001] The present invention relates to a polypeptide for delaying telomere shortening and application thereof, belonging to the field of biotechnology. Background Art

[0002] Telomeres are the natural ends of linear chromosomes in eukaryotic organisms, complex structures formed by repetitive DNA sequences and proteins. Telomeres maintain chromosomal stability and integrity, preventing chromosome degradation, fusion, recombination, and loss. They also protect against damage to chromosome ends by intracellular exonucleases, endonucleases, ligases, topoisomerases, and proteases, thereby maintaining the stability of the genetic system. Telomeres not only maintain chromosomal integrity but also serve as key regulators of cell lifespan.

[0003] Telomere shortening is considered a molecular clock that triggers cellular aging and can serve as a biomarker of aging. Studies have shown that normal human tissues and cells experience telomeric DNA loss during replication. During cell division in vivo or in vitro, normal somatic cells lose 50 to 200 base pairs of telomere DNA with each cell division. When telomeres shorten to a certain length, cells lose their ability to divide and enter a state of senescence. Telomere loss or dysfunction can lead to a variety of pathological conditions, including cell proliferation failure, apoptosis, and even the induction of serious diseases such as genetic instability and cancer. Therefore, the length and function of telomeres are crucial for maintaining cellular health and preventing disease. The discovery of drugs that can effectively slow telomere shortening would be highly beneficial for combating aging and treating diseases associated with telomere loss or dysfunction. Summary of the Invention

[0004] To solve the above problems, the present invention provides a polypeptide for delaying telomere shortening, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0005] The present invention also provides a polypeptide composition for delaying telomere shortening, wherein the components of the polypeptide composition include the above-mentioned polypeptide and a polypeptide having an amino acid sequence as shown in SEQ ID NO.2.

[0006] In one embodiment of the present invention, in the polypeptide composition, the mass ratio of the polypeptide having an amino acid sequence as shown in SEQ ID NO.1 to the polypeptide having an amino acid sequence as shown in SEQ ID NO.2 is 1-4:1-4.

[0007] The present invention also provides a nucleic acid molecule encoding the above polypeptide.

[0008] The present invention also provides a recombinant plasmid, which expresses the polypeptide.

[0009] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.

[0010] In one embodiment of the present invention, the plasmid vector includes pET-GST plasmid, pET-His plasmid, pllp-STII plasmid and / or pMBP-C plasmid.

[0011] In one embodiment of the present invention, the method for preparing the recombinant plasmid is: inserting the above-mentioned nucleic acid molecule into a vector to obtain the recombinant plasmid.

[0012] The present invention also provides a host cell, wherein the genome of the host cell is integrated with the above nucleic acid molecule; or the host cell carries the above recombinant plasmid.

[0013] In one embodiment of the present invention, the host cell comprises a fungus, a bacterium, a plant cell and / or an animal cell.

[0014] The present invention also provides the use of the above polypeptide, the above polypeptide composition, the above nucleic acid molecule, the above recombinant plasmid, or the above host cell in the preparation of a drug, wherein the drug has any of the following functions:

[0015] (a) Delaying telomere shortening;

[0016] (b) anti-aging;

[0017] and / or, (c) preventing and / or treating diseases associated with telomere loss or dysfunction.

[0018] In one embodiment of the present invention, in the drug, the mass ratio of the polypeptide having an amino acid sequence as shown in SEQ ID NO.1 to the polypeptide having an amino acid sequence as shown in SEQ ID NO.2 is 1-4:1-4.

[0019] In one embodiment of the present invention, the anti-aging comprises delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase and / or inhibiting the expression level of β-galactosidase to resist aging;

[0020] The prevention and / or treatment of diseases associated with telomere loss or dysfunction includes preventing and / or treating diseases associated with telomere loss or dysfunction by delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase and / or inhibiting the expression level of β-galactosidase.

[0021] In one embodiment of the present invention, the diseases associated with telomere loss or dysfunction include neurodegenerative diseases, liver cirrhosis and / or renal damage; the neurodegenerative diseases include Alzheimer's disease and / or Parkinson's disease.

[0022] In one embodiment of the present invention, the ingredients of the drug further include pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include maltodextrin, gum arabic and / or soluble starch.

[0023] In one embodiment of the present invention, the disease of the medicine comprises tablets, capsules, granules, sustained-release preparations and / or injections.

[0024] The present invention also provides a drug, the components of which include the above-mentioned polypeptide, the above-mentioned polypeptide combination, the above-mentioned nucleic acid molecule, the above-mentioned recombinant plasmid and / or the above-mentioned host cell;

[0025] The drug has any of the following functions:

[0026] (a) Delaying telomere shortening;

[0027] (b) anti-aging;

[0028] and / or, (c) preventing and / or treating diseases associated with telomere loss or dysfunction.

[0029] In one embodiment of the present invention, in the drug, the mass ratio of the polypeptide having an amino acid sequence as shown in SEQ ID NO.1 to the polypeptide having an amino acid sequence as shown in SEQ ID NO.2 is 1-4:1-4.

[0030] In one embodiment of the present invention, the anti-aging comprises delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase and / or inhibiting the expression level of β-galactosidase to resist aging;

[0031] The prevention and / or treatment of diseases associated with telomere loss or dysfunction includes preventing and / or treating diseases associated with telomere loss or dysfunction by delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase and / or inhibiting the expression level of β-galactosidase.

[0032] In one embodiment of the present invention, the diseases associated with telomere loss or dysfunction include neurodegenerative diseases, liver cirrhosis and / or renal damage; the neurodegenerative diseases include Alzheimer's disease and / or Parkinson's disease.

[0033] In one embodiment of the present invention, the ingredients of the drug further include pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include maltodextrin, gum arabic and / or soluble starch.

[0034] In one embodiment of the present invention, the disease of the medicine comprises tablets, capsules, granules, sustained-release preparations and / or injections.

[0035] The technical solution of the present invention has the following advantages:

[0036] 1. The present invention provides a polypeptide for delaying telomere shortening, the amino acid sequence of which is shown in SEQ ID NO. 1. Studies have shown that the polypeptide has highly effective biological activity, significantly slowing the cellular aging process by delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing telomerase activity, and inhibiting the expression level of β-galactosidase. Furthermore, the polypeptide is derived from the fermentation process of natural dairy products, offering high safety and a low risk of side effects. Furthermore, the polypeptide has a small molecular weight and is easily absorbed by the human body, which can improve drug bioavailability. Therefore, the polypeptide has great application prospects in delaying telomere shortening, anti-aging, and the treatment of diseases associated with telomere loss or dysfunction.

[0037] 2. The present invention provides a polypeptide composition for delaying telomere shortening, comprising a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide having an amino acid sequence as shown in SEQ ID NO.2. Studies have shown that the polypeptide composition has highly effective biological activity and can significantly slow the cellular aging process by delaying telomere shortening, increasing the expression level of telomerase reverse transcriptase, increasing the activity of telomerase, and inhibiting the expression level of β-galactosidase. Furthermore, both polypeptides in the polypeptide composition are derived from the fermentation process of natural dairy products, thus having high safety and low risk of side effects. Furthermore, the two polypeptides in the polypeptide composition have a low average molecular weight, are easily absorbed by the human body, and can improve the bioavailability of the drug. Therefore, the polypeptide composition has great application prospects in delaying telomere shortening, anti-aging, and the treatment of diseases related to telomere loss or dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Relative telomere length of leukocytes in peripheral blood of different groups of mice.

[0039] Figure 2 : The expression levels of telomerase (TE) in the livers of mice in different groups.

[0040] Figure 3 : The expression levels of telomerase reverse transcriptase (TERT) in the livers of mice in different groups.

[0041] Figure 4 : The number of β-galactosidase (β-gal) blue positive signal particles in the livers of mice in different groups.

[0042] Figures 1 to 4 In the table, # indicates P < 0.05 compared with the blank group, and * indicates P < 0.05 compared with the model group. DETAILED DESCRIPTION

[0043] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0044] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0045] Example 1: A polypeptide for delaying telomere shortening

[0046] This embodiment provides a polypeptide for delaying telomere shortening, the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 (LDAYPSG) (the polypeptide is derived from a fermented dairy product of a standard strain of Lactobacillus bulgaricus, the Genbank sequence number of the standard strain of Lactobacillus bulgaricus is CP032451.1).

[0047] Example 2: A polypeptide composition for delaying telomere shortening

[0048] This embodiment provides a polypeptide composition for delaying telomere shortening, the polypeptide composition consisting of a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 (LDAYPSG) and a polypeptide having an amino acid sequence as shown in SEQ ID NO.2 (VLNPW), and in the polypeptide composition, the mass ratio of the polypeptide having an amino acid sequence as shown in SEQ ID NO.1 (LDAYPSG) to the polypeptide having an amino acid sequence as shown in SEQ ID NO.2 (VLNPW) is 1:1.

[0049] Comparative Example 1: A polypeptide for delaying telomere shortening

[0050] This comparative example provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 2 (VLNPW) (the polypeptide is also derived from a fermented dairy product of a standard strain of Lactobacillus bulgaricus, and the Genbank sequence number of the standard strain of Lactobacillus bulgaricus is CP032451.1).

[0051] Comparative Example 2: A polypeptide for delaying telomere shortening

[0052] This comparative example provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 3 (QGPIVLNPWDQVK) (the polypeptide is also derived from a fermented dairy product of a standard strain of Lactobacillus bulgaricus, and the Genbank sequence number of the standard strain of Lactobacillus bulgaricus is CP032451.1).

[0053] Experimental Example 1: Functional Verification of Peptides for Delaying Telomere Shortening

[0054] This experimental example provides a functional validation experiment for a peptide used to delay telomere shortening. The experimental process is as follows:

[0055] 1. Experimental methods

[0056] Sixty two-month-old SPF-grade male C57BL / 6 mice (weighing 22-26 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were randomly divided into six groups (10 mice per group) after one week of adaptive feeding to begin the experiment. The six groups were blank control group, model group, Example 1 group, Example 2 group, Comparative Example 3 group, and Comparative Example 4 group, with 10 mice per group. Each group of mice was raised in a clean environment with constant temperature and humidity (temperature 22-25°C, humidity 65-70%), with 12 hours of light and 12 hours of dark alternating day and night, and in a well-ventilated environment with free access to food and water.

[0057] After one week of adaptive feeding, the aging model was established by intraperitoneal injection of 80 mg / kg D-galactose solution (10 mg / mL, prepared in normal saline) into the five groups of mice except the blank control group, which was intraperitoneally injected with an equal volume of normal saline for 7 consecutive weeks. Intervention began on the 8th week, and the intervention scheme for each group of mice was as follows: the mice in Group 1 of Example were gavaged with a LDAYPSG + VLNPW polypeptide combination solution at a dose of 20 mL / kg / d (in the solution, the concentration of LDAYPSG was 15 mg / mL, the concentration of VLNPW was 15 mg / mL, and the solvent was normal saline), the mice in Group 2 of Example were gavaged with a LDAYPSG polypeptide solution at a dose of 20 mL / kg / d (in the solution, the concentration of LDAYPSG was 15 mg / mL, and the solvent was normal saline), the mice in Group 3 of Comparative Example were gavaged with a VLNPW polypeptide solution at a dose of 20 mL / kg / d (in the solution, the concentration of LDAYPSG was 15 mg / mL, and the solvent was normal saline), the mice in Group 4 of Comparative Example were gavaged with a QGPIVLNPWDQVK polypeptide solution at a dose of 20 mL / kg / d (in the solution, the concentration of QGPIVLNPWDQVK was 15 mg / mL, and the solvent was normal saline), and the mice in the model group and the control group were gavaged with an equal amount of sterile saline for 8 weeks.

[0058] 24 hours after the last oral administration, the eyes of mice in each group were removed and blood was collected before being sacrificed. The livers of mice in each group were collected and divided into two parts. One part was immediately frozen in liquid nitrogen and then transferred to -80°C for storage. The other part was fixed with paraformaldehyde for subsequent experimental testing. The testing process was as follows:

[0059] Telomeres are considered to be the clock of cell division, and telomere length is an important marker of aging. The qPCR method was used to detect the relative telomere length of leukocytes in each group of mice using 36B4 as an internal reference to examine the effects of each intervention on the telomere length of mice. The test results are shown in Figure 1 ; The primers used for qPCR are shown in Table 1.

[0060] Telomere attrition is an important factor leading to aging, and the maintenance of telomere length mainly depends on the activity of telomerase (TE). Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and plays an important role in the regulation of telomerase activity. It can regulate the expression of telomerase at the transcriptional and translational levels. A PCR-ELISA kit (purchased from Roche, Switzerland) was used to detect the telomerase (TE) activity in mouse liver, and PCR was used to detect the mRNA expression of telomerase reverse transcriptase (TERT) to test the effects of each group of interventions on the telomerase activity and telomerase reverse transcriptase expression levels in mice. The test results are shown in Figures 2 and 3 ; The primers used for qPCR are shown in Table 1.

[0061] Aging-related β-galactosidase (β-galactosidase, β-gal) is a potential marker of cellular aging. When cells or tissues undergo aging, the expression of β-galactosidase increases significantly, and its activity is positively correlated with the number of blue-positive signal particles after staining. The liver tissues of each group of mice were fixed with paraformaldehyde and first soaked in sucrose overnight (16 hours) before preparing frozen sections. The sections were then developed with the incubation solution of the β-galactosidase detection kit (purchased from CST, USA) overnight (16 hours). After counterstaining with neutral red, the number of blue-positive signal particles in each group of sections was observed under an oil immersion lens (the number of blue-positive signal particles represents the expression level of β-galactosidase) to test the effect of each intervention group on the expression level of β-galactosidase in mice. The test results are shown in the table. Figure 4 .

[0062] Table 1 qPCR primer sequences

[0063]

[0064]

[0065] 2. Experimental results

[0066] The results of relative telomere length detection of mouse peripheral blood leukocytes are as follows Figure 1 As shown, the telomere length of the model group decreased significantly, with a significant difference from the blank control group (P<0.05); the relative telomere length of the Example 1 group was higher than that of the model group, and the difference was significant (P<0.05); the relative telomere length of the Example 2 group and the Comparative Example 3 group was slightly higher than that of the model group, but lower than that of the Example 1 group; and the relative telomere length of the Comparative Example 4 group was not significantly different from that of the model group. These results show that the Example 1 group, the Example 2 group, and the Comparative Example 3 group have the effect of protecting telomere length.

[0067] The results of mouse telomerase (TE) and telomerase reverse transcriptase (TERT) detection are as follows Figures 2 and 3 As shown, the TE and TERT levels in the model group were lower than those in the blank control group, and the difference was significant (P<0.05); the TE and TERT levels in the Example 1 group were higher than those in the model group, and the difference was significant (P<0.05); the TE level in the Example 2 group was slightly higher than that in the model group, and the TERT level in the Example 2 group was significantly different from that in the model group, but both were lower than that in the Example 1 group; while the TE and TERT levels in the Comparative Example 3 and Comparative Example 4 groups were not significantly different from those in the model group. These results indicate that the Example 1 and Example 2 groups were able to increase the expression levels of TE and TERT and improve telomerase activity, with significant effects.

[0068] The results of mouse liver β-gal are as follows Figure 4As shown, the number of positive signal particles in the model group was much higher than that in the blank control group, and the difference was significant (P < 0.05); the number of blue positive signal particles in Example 1 group was higher than that in the model group, and the difference was significant (P < 0.05); the number of blue positive signal particles in Example 2 group was also significantly different from that in the model group, but lower than that in Example 1 group; while the number of blue positive signal particles in Comparative Example 3 and Comparative Example 4 groups was not significantly different from that in the model group. These results indicate that Example 1 and Example 2 groups have an inhibitory effect on β-gal in mouse livers and can slow down aging to a certain extent.

[0069] comprehensive Figures 1 to 4 The results show that the experimental mouse model was successfully established, and the intervention of Example 1 and Example 2 groups can increase telomerase activity, delay telomere attrition, protect telomeres, and reduce the expression of aging-related β-galactosidase, thereby delaying aging. Among them, the effect of delaying aging in Example 1 group is the most significant, followed by Example 2 group.

[0070] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polypeptide composition for delaying telomere shortening, characterized in that: The components of the polypeptide composition include a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide with an amino acid sequence as shown in SEQ ID NO.2; in the polypeptide composition, the mass ratio of the polypeptide with an amino acid sequence as shown in SEQ ID NO.1 to the polypeptide with an amino acid sequence as shown in SEQ ID NO.2 is 1-4:1-4.

2. Use of the polypeptide composition according to claim 1 in preparing a medicine, characterized in that: The drug has any of the following functions: (a) Anti-aging; and / or, (b) preventing and / or treating diseases associated with telomere loss or dysfunction; The disease associated with telomere loss or dysfunction is a neurodegenerative disease, liver cirrhosis and / or kidney damage; the neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease.

3. A drug, characterized in that The composition of the medicine comprises the polypeptide composition of claim 1; The drug has any of the following functions: (a) Anti-aging; and / or, (b) preventing and / or treating diseases associated with telomere loss or dysfunction; The disease associated with telomere loss or dysfunction is a neurodegenerative disease, liver cirrhosis and / or kidney damage; the neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease.

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