Polypeptide for delaying telomere shortening and application thereof

Abstract

The invention relates to a polypeptide for delaying telomere shortening and application thereof, and belongs to the technical field of biology. The invention provides a polypeptide for delaying telomere shortening. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. Researches show that the polypeptide has efficient biological activity, and can significantly slow down the cell aging process by delaying telomere shortening, improving the expression level of telomerase reverse transcriptase, improving the activity of telomerase and inhibiting the expression level of beta-galactosidase. Moreover, the polypeptide is derived from a fermentation process of a natural dairy product, and has relatively high safety and relatively low side effect risk. In addition, the polypeptide is small in molecular weight and easy to absorb by the human body, and the bioavailability of the medicine can be improved. Therefore, the polypeptide has great application prospects in delaying telomere shortening, resisting aging and treating diseases related to telomere deletion or dysfunction.

Description

Technical Field

[0001] The 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, and are complex structures formed by the combination of repeated DNA sequences and proteins. Telomeres have the function of maintaining chromosome stability and integrity, preventing chromosome degradation, fusion, recombination and loss, resisting damage to chromosome ends by exonucleases, endonucleases, ligases, topoisomerases and proteases in cells, and maintaining the stability of the genetic system. Telomeres not only maintain the integrity of chromosomes, but also exist as key regulatory factors for cell survival.

[0003] Telomere shortening is considered to be the molecular clock that triggers cell aging and can be used as a biomarker of aging. Studies have shown that normal human tissues and cells lose telomere DNA during replication. During the division of normal somatic cells in vivo or in vitro, telomere DNA decreases by 50 to 200 bp each time the cells divide. When the telomere shortens to a certain length, the cell loses the ability to divide and enters a state of aging. Telomere loss or dysfunction will lead to a variety of pathological conditions, including cell proliferation failure, cell apoptosis, and even induce serious diseases such as genetic instability and cancer. Therefore, the length and functional state of telomeres are of great significance for maintaining cell health and preventing the occurrence of diseases. If a drug that can effectively delay telomere shortening can be found, it will be very helpful for the treatment of anti-aging and diseases related to telomere loss or dysfunction. Summary of the invention

[0004] In order to solve the above problems, 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.

[0005] The present invention also provides a polypeptide composition for delaying telomere shortening, wherein the components of the polypeptide composition include the above 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, which encodes the above polypeptide.

[0008] The present invention also provides a recombinant plasmid, which expresses the above 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 comprises 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 nucleic acid molecule into a vector to obtain the recombinant plasmid.

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

[0013] In one embodiment of the present invention, the host cell comprises fungi, bacteria, plant cells and / or animal cells.

[0014] The present invention also provides the use of the above polypeptide or the above polypeptide composition or the above nucleic acid molecule or 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) Slow down 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 injury; 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 diseases of the medicine include tablets, capsules, granules, sustained-release preparations and / or injections.

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

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

[0026] (a) Slow down 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 injury; 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 diseases of the medicine include 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, 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, is easily absorbed by the human body, and 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.

[0037] 2. The present invention provides a polypeptide composition for delaying telomere shortening, wherein the components of the polypeptide composition include 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 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. Moreover, both polypeptides of the polypeptide composition are derived from the fermentation process of natural dairy products, and have high safety and low risk of side effects. In addition, the two polypeptides of the polypeptide composition have a small 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 : Expression levels of telomerase (TE) in the livers of mice in different groups.

[0040] Figure 3 : The expression level of telomerase reverse transcriptase (TERT) in the liver 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] Figure 1 to Figure 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, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0044] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[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, and 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, wherein the polypeptide composition is composed 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) and 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 verification experiment of a peptide for delaying telomere shortening. The experimental process is as follows:

[0055] 1. Experimental methods

[0056] 60 2-month-old SPF male C57BL / 6 mice (weight 22-26g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were randomly divided into 6 groups (10 mice in each group) after one week of adaptive feeding to start the experiment. The 6 groups were blank control group, model group, Example 1 group, Example 2 group, Comparative Example 3 group and Comparative Example 4 group, 10 mice in each group. Each group of mice was raised in a clean environment with constant temperature and humidity (temperature 22-25°C, humidity 65-70%), and maintained with 12h light and 12h dark day and night alternation, and raised in a well-ventilated environment with free 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 5 groups of mice except the blank control group, which was intraperitoneally injected with an equal volume of normal saline for 7 consecutive weeks. The intervention started at week 8, 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 normal saline for 8 weeks.

[0058] 24 hours after the last oral administration, the eyeballs of mice in each group were removed and blood was collected and then killed. 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℃ for storage, and the other part was fixed with paraformaldehyde for subsequent experimental detection. The detection process is 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 with 36B4 as the internal reference to examine the effect of each group of intervention on the telomere length of mice. The test results are shown in Figure 1 ; Among them, 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. The telomerase (TE) activity in mouse liver was detected by PCR-ELISA kit (purchased from Roche, Switzerland), and the mRNA expression of telomerase reverse transcriptase (TERT) was detected by PCR to examine the effects of each group of interventions on the telomerase activity and telomerase reverse transcriptase expression level of mice. The test results are shown in Figure 2-Figure 3 ; Among them, the primers used for qPCR are shown in Table 1.

[0061] Aging-related β-galactosidase (β-galactosidase, β-gal) is a potential marker of cell aging. When cells or tissues age, 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, soaked in sucrose overnight (16 hours) and then prepared for frozen sections. Then, the incubation solution of the β-galactosidase detection kit (purchased from CST, USA) was used for color development overnight (16 hours). After neutral red counterstaining, the number of blue positive signal particles in each group of sections was observed under an oil microscope (the number of blue positive signal particles represents the expression of β-galactosidase) to test the effect of each group of interventions on the expression level of β-galactosidase in mice. The test results are shown in Figure 4 .

[0062] Table 1 qPCR primer sequences

[0063]

[0064]

[0065] 2. Experimental results

[0066] The results of relative telomere length test of mouse peripheral blood leukocytes are as follows Figure 1 As shown, the telomere length of the model group decreased significantly, which was significantly different 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. This result shows that the Example 1 group, the Example 2 group and the Comparative Example 3 group have the effect of protecting the telomere length.

[0067] The results of mouse telomerase (TE) and telomerase reverse transcriptase (TERT) detection are as follows Figure 2-Figure 3 As shown, the TE and TERT levels in the model group were lower than those in the blank control group, and there was a significant difference (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. This result shows that the Example 1 and Example 2 groups can increase the expression levels of TE and TERT and improve telomerase activity, with significant effects.

[0068] Mouse liver β-gal results are as follows Figure 4As shown, the number of positive signal particles in the model group is much higher than that in the blank control group, and the difference is significant (P<0.05); the number of blue positive signal particles in Example 1 group is higher than that in the model group, and there is a significant difference (P<0.05); the number of blue positive signal particles in Example 2 group is 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 is not significantly different from that in the model group. This result shows that Example 1 and Example 2 groups have an inhibitory effect on β-gal in mouse liver and can slow down aging to a certain extent.

[0069] comprehensive Figure 1 to Figure 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 wear, 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 the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the 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 the preparation of medicines, characterized in that: The drug has any of the following functions: (a) Slow down telomere shortening; (b) Anti-aging; and / or, (c) preventing and / or treating diseases associated with telomere loss or dysfunction.

3. The use according to claim 2, characterized in that The anti-aging method includes 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; 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.

4. A drug, characterized in that The composition of the drug comprises the polypeptide composition of claim 1; The drug has any of the following functions: (a) Slow down telomere shortening; (b) Anti-aging; and / or, (c) preventing and / or treating diseases associated with telomere loss or dysfunction.

5. The drug according to claim 4, characterized in that The anti-aging method includes 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; 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.

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