A neurotrophic composition for aiding sleep and relieving depression

Abstract

The present invention provides a protein comprising an amino acid sequence as shown in SEQ ID NO: 1 or 3. Also provided are corresponding nucleic acids, vectors, and compositions comprising the protein. In some embodiments, the composition comprises a recombinant collagen-like protein having an amino acid sequence as shown in SEQ ID NO: 1 or 3, γ-aminobutyric acid, calcium gluconate, zinc gluconate, vitamins, oryzanol, a recombinant humanized collagen having an amino acid sequence as shown in SEQ ID NO: 4, and cordyceps powder.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a protein, a composition thereof, and uses thereof. Background Art

[0002] Insufficient sleep is common in modern society and can be due to a variety of factors, such as work pressures, family responsibilities, physical illness, and sleep disorders. As sleep deprivation accumulates, individuals may experience reduced performance, increased risk of accidents, and death. Sleep is measured in two ways: duration and depth. When individuals do not get enough sleep or a good quality of sleep, daytime wakefulness and cognitive function are impaired. It is often difficult to determine the amount of sleep that is normal for a particular individual. In general, sleep needs vary significantly between individuals and at different stages of life. Some individuals may report needing 10 hours or more of sleep per night. About a third of adults report sleeping less than 7 hours per night on weekdays. Short sleep duration also varies by occupation and industry.

[0003] A meta-analysis of more than 690,000 children in 20 countries found that children's sleep duration has decreased by 0.75 minutes per year over the past 100 years; the rate of change was greatest during school years, among older children, and among males.

[0004] Lack of sleep can cause various harms.

[0005] Experiments have shown that the most prominent effect of total sleep deprivation and consecutive nights of sleep restriction is cognitive impairment. Sleeping less than 7 hours a night can lead to cumulative deficits in behavioral alertness and vigilant attention. Even a few hours of sleep loss can affect individuals' ability to complete tasks that require sustained concentration.

[0006] Sleep deprivation can also lead to a mental state similar to depression or anxiety, with patients reporting bad mood, irritability, decreased energy, loss of libido, poor judgment, and other signs of psychological dysfunction. The cumulative damage from long-term sleep deprivation is often difficult to make up for. Attention deficits caused by sleep loss can be detected in the laboratory using special quantitative tests. For example, an individual's reaction time to visual stimuli is recorded. Sleep deprivation can cause an overall slowing of reaction time and a steady increase in the number of omission errors (i.e., inattention).

[0007] A series of representative prospective studies have demonstrated the association between insufficient sleep and various adverse outcomes: it is independently associated with an increased risk of multiple cardiovascular metabolic risk factors, including central obesity (HR 1.12), elevated fasting blood glucose (HR 1.06), hypertension (HR 1.08), decreased high-density lipoprotein (HR 1.07), hypertriglyceridemia (HR 1.09), and metabolic syndrome (HR 1.09); it increases the levels of inflammatory markers and causes immune deficiency; it increases susceptibility to the common cold; it increases the risk of developing metabolic syndrome (such as obesity) by up to 3 times; it affects appetite and hormones (such as leptin and appetite-stimulating hormone), leading to weight gain and the risk of diabetes; and it increases all-cause mortality.

[0008] It is reported that if you sleep less than 6 hours, your risk of depression may increase by 80%. The National Sleep Foundation recommends that people sleep 7 to 9 hours a night. Even if you sleep one hour less than the minimum recommended sleep time, it is easy to make people feel nervous, helpless or uneasy.

[0009] There is currently an urgent need for products that help with sleep and relieve depression. Summary of the invention

[0010] After in-depth research, design, screening and experimental verification, the inventors obtained a recombinant collagen-like protein (fusion factor PHPV1.6), and the recombinant protein and the neurotrophic composition containing it have obvious effects of helping sleep and relieving depression. Therefore, the present invention was obtained.

[0011] In a first aspect of the present invention, a protein is provided, characterized in that the protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or 3.

[0012] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 1 or 3.

[0014] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 1. In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 3.

[0015] In some embodiments of the present invention, the method for preparing the protein is as described in Example 1.

[0016] In the second aspect of the present invention, a nucleic acid is provided, characterized in that the nucleic acid encodes the protein as described in any one of the first aspect.

[0017] In some embodiments of the present invention, a nucleic acid is provided, characterized in that the nucleic acid encodes a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 3.

[0018] In some embodiments of the invention, the nucleic acid encodes a protein whose amino acid sequence is shown in SEQ ID NO: 1. In some embodiments of the invention, the nucleic acid encodes a protein whose amino acid sequence is shown in SEQ ID NO:3.

[0019] In some embodiments, the coding sequence is codon optimized.

[0020] In some embodiments, the codons are optimized for expression in eukaryotic or prokaryotic cells.

[0021] In some embodiments, the prokaryotic cell is Escherichia coli, Bacillus subtilis, or Bacillus licheniformis.

[0022] In some embodiments, the eukaryotic cell is a Pichia pastoris, Saccharomyces cerevisiae, a CHO cell, or a HEK293 cell.

[0023] In the third aspect of the present invention, a vector is provided, characterized in that the vector comprises a nucleotide sequence encoding the protein as described in the first aspect, and a promoter sequence connected to the nucleotide sequence.

[0024] In some embodiments of the present invention, a vector is provided, characterized in that the vector comprises a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 3.

[0025] In some embodiments, the vector comprises a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the vector comprises a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 3.

[0026] In some embodiments, the vector is a plasmid.

[0027] In a fourth aspect of the present invention, a cell is provided, characterized in that the cell comprises the nucleic acid as described in the second aspect or the vector as described in the third aspect.

[0028] In the fifth aspect of the present invention, a composition is provided, characterized in that the composition comprises the protein as described in the first aspect.

[0029] In some embodiments of the present invention, a composition is provided, characterized in that the composition comprises a protein with an amino acid sequence as shown in SEQ ID NO: 1 or 3.

[0030] In some embodiments, the composition comprises a protein having an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the composition comprises a protein having an amino acid sequence as shown in SEQ ID NO:3.

[0031] In some embodiments, the composition comprises a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 3, γ-aminobutyric acid, calcium gluconate, zinc gluconate, vitamins, oryzanol, a protein having an amino acid sequence as shown in SEQ ID NO: 4, and Cordyceps sinensis powder.

[0032] In some embodiments, the composition comprises a protein with an amino acid sequence as shown in SEQ ID NO: 1, γ-aminobutyric acid, calcium gluconate, zinc gluconate, vitamins, oryzanol, a protein with an amino acid sequence as shown in SEQ ID NO: 4, and Cordyceps sinensis powder.

[0033] In some embodiments, the composition comprises 0.8-1.2 parts by weight of a protein having an amino acid sequence as shown in SEQ ID NO: 1, 4-6 parts by weight of γ-aminobutyric acid, 4-6 parts by weight of calcium gluconate, 4-6 parts by weight of zinc gluconate, 0.8-1.2 parts by weight of vitamin B1, 0.8-1.2 parts by weight of vitamin B2, 0.8-1.2 parts by weight of vitamin B6, 0.8-1.2 parts by weight of niacin, 0.8-1.2 parts by weight of oryzanol, 0.8-1.2 parts by weight of a protein having an amino acid sequence as shown in SEQ ID NO: 4, and 0.8-1.2 parts by weight of Cordyceps sinensis powder.

[0034] In some embodiments, the composition comprises 0.8-1.2 parts by weight of a protein having an amino acid sequence as shown in SEQ ID NO:3, 4-6 parts by weight of γ-aminobutyric acid, 4-6 parts by weight of calcium gluconate, 4-6 parts by weight of zinc gluconate, 0.8-1.2 parts by weight of vitamin B1, 0.8-1.2 parts by weight of vitamin B2, 0.8-1.2 parts by weight of vitamin B6, 0.8-1.2 parts by weight of niacin, 0.8-1.2 parts by weight of oryzanol, 0.8-1.2 parts by weight of a protein having an amino acid sequence as shown in SEQ ID NO:4, and 0.8-1.2 parts by weight of Cordyceps sinensis powder.

[0035] In some embodiments, the composition comprises 1 part by weight of a protein having an amino acid sequence as shown in SEQ ID NO: 1, 5 parts by weight of γ-aminobutyric acid, 5 parts by weight of calcium gluconate, 5 parts by weight of zinc gluconate, 1 part by weight of vitamin B1, 1 part by weight of vitamin B2, 1 part by weight of vitamin B6, 1 part by weight of niacin, 1 part by weight of oryzanol, 1 part by weight of a protein having an amino acid sequence as shown in SEQ ID NO: 4, and 1 part by weight of Cordyceps sinensis powder.

[0036] In some embodiments, the composition comprises 1 part by weight of a protein having an amino acid sequence as shown in SEQ ID NO:3, 5 parts by weight of γ-aminobutyric acid, 5 parts by weight of calcium gluconate, 5 parts by weight of zinc gluconate, 1 part by weight of vitamin B1, 1 part by weight of vitamin B2, 1 part by weight of vitamin B6, 1 part by weight of niacin, 1 part by weight of oryzanol, 1 part by weight of a protein having an amino acid sequence as shown in SEQ ID NO:4, and 1 part by weight of Cordyceps sinensis powder.

[0037] In some embodiments of the present invention, the preparation method of the composition is as described in Example 2.

[0038] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. DETAILED DESCRIPTION

[0039] The present disclosure is further illustrated by way of examples below, but the present disclosure is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions. Unless otherwise specified, the reagents and raw materials used in the present disclosure are all commercially available.

[0040] Example 1: Design and preparation of recombinant protein

[0041] The recombinant collagen-like protein (named as fusion factor PHPV1.6) with a sequence as shown in SEQ ID NO: 1 was designed. In a non-limiting manner, it is obtained by fusing a mutant human collagen domain, a mature fragment of a brain-derived neurotrophic factor, a linker, a mutant lactoferrin biodefense functional peptide, and a his tag. In addition, an enterokinase recognition cleavage site is added so that the recombinant protein can be enzymatically cleaved into multiple different functional peptides in the stomach or small intestine.

[0042] The amino acid sequence of the recombinant collagen-like protein (fusion factor PHPV1.6) is:

[0043] (SEQ ID NO: 1).

[0044] The nucleic acid fragment encoding the recombinant collagen-like protein synthesized outsourced (codon optimized for E. coli) was cloned between the BlpI and XbaI restriction sites of the pET28a(+) vector. The reaction solution was transformed into stbl3 competent cells, cultured for 18 hours, and then the positive clones were selected for sequencing for identification. After the positive clones with correct sequencing were cultured, the expression vector plasmid of the recombinant collagen-like protein (SEQ ID NO: 2) was extracted.

[0045] The obtained vector plasmid was transformed into BL21 (DE3) E. coli competent cells (NEB, C2527I). Cultured in LB culture medium containing kanamycin at 37°C for 17 hours. Add IPTG to a concentration of 0.5mM and induce at 16°C for 25 hours. Centrifuge and collect the bacteria, add lysis solution and ultrasonically break them, and centrifuge again to obtain the supernatant containing recombinant collagen-like protein. After filtering with a 0.45μm filter membrane, purify it with Ni-NTA affinity chromatography, and then use ion chromatography chromatography, and freeze-dry the eluate to obtain a recombinant collagen-like protein with an HPLC purity of 97.1%, that is, the fusion factor PHPV1.6 (SEQ ID NO: 1). This sample only presents a single band in SDS-PAGE.

[0046] The same method was used to prepare the non-mutated recombinant collagen FLM1.6.0 (SEQ ID NO: 3):

[0047] .

[0048] The same method was used to prepare recombinant humanized collagen (Fuhuo Factor) FHTFF1.6 (SEQ ID NO: 4):

[0049] MGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGA EGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGRGVVGLPGPQGIAGQRGVVGLPGERGERGER(SEQ ID NO:4).

[0050] Example 2: Preparation of the composition

[0051] The recombinant collagen fusion factor PHPV1.6 (100 mg) prepared by the method described in Example 1 is mixed with γ-aminobutyric acid (500 mg), calcium gluconate (500 mg), zinc gluconate (500 mg), multiple vitamins (vitamin B1 100 mg, vitamin B2 100 mg, vitamin B6 100 mg, niacin 100 mg, oryzanol 100 mg), the Fuhuo factor FHTFF1.6 (100 mg) prepared by the method described in Example 1, and cordyceps powder (Psaitong, product number C14444, 100 mg) to obtain a solid powder composition 1.

[0052] The same method was used to prepare a solid powder composition 2 by replacing the recombinant collagen fusion factor PHPV1.6 with an equal mass of the protein FLM1.6.0 prepared as described in Example 1.

[0053] Example 3: Effect test of recombinant collagen and its composition

[0054] Take healthy 4-week-old SPF-grade ICR male mice and ensure that the mice have enough feed and clean water every day. The lighting conditions are 12 hours of light and 12 hours of darkness every day (lights are turned on from 9 am to 21 pm every day), the room temperature is maintained at 22-25°C, and the humidity is maintained at 55%±10%. After acclimatization for 5 days, the body weight was measured to be 21.1-22.0g. Randomly grouped, 12 animals in each group. Including blank control group, model group, positive control equol group, and 3 experimental groups (including composition group 1, composition group 2, and recombinant collagen group).

[0055] Then, on the second day after the adaptation to the environment, the mice in other groups, except the blank control group, were placed in the drum of the rat and mouse sleep disruption instrument (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.) for adaptation; the parameters were set as follows: the drum speed was 1 rpm, and the animals rested for 2 minutes after each rotation, and maintained for 3 hours a day. The animals were continuously adapted to the drum for 3 days, and the animals were placed back in the cages after the adaptation was completed each day.

[0056] Then, on the second day after the adaptation to the drum, the model was started: a sleep interference device (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.) was used for interference every day; the setting parameters were: the drum speed was 1 rpm, and there was a 2-minute rest after each rotation, which was maintained for 3 hours a day (from 13:00 to 16:00 in the afternoon) for 15 consecutive days.

[0057] During the modeling period, the mice were taken out of the drum apparatus every day and gavaged with 0.2 mL of the corresponding test substance (the dose was 20 mg of the test substance / kg body weight), and then quickly put back into the drum to continue sleep interference. At the same time, the blank control group and the model group mice were kept in the cage and received the same volume of water, and the positive control group was gavaged with 0.2 mL of the equol aqueous dispersion (the dose was 20 mg of equol / kg body weight). The administration was continued for 15 days.

[0058] The mice were weighed on the first day of modeling and on the second day after modeling.

[0059] Mouse tail suspension experiment: On the second day after the modeling was completed, the mouse tail was wrapped with tape about 1 cm away from the tip of the tail before the experiment began, and the mouse was hung upside down on an iron hook. It was first allowed to adapt for 2 minutes, and then the small animal behavior tracking system (Noldus) was used to automatically record the mouse's immobility time in the following 4 minutes.

[0060] Mouse forced swimming test: On the second day after the tail suspension test, 5 hours after the tail suspension test, the mice were placed in a mouse constant temperature swimming pool (Beijing Zhishu Duobao), and the water temperature was maintained at 23-25°C. First, they were allowed to adapt for 2 minutes, and then the immobility time of the mice was recorded within the next 4 minutes.

[0061] The experimental results are shown in Table 1. The result data are the average values ​​of the animals in each group. The model was successfully established.

[0062] The weight gain on the 15th day of modeling compared to the 1st day of modeling was higher in the positive control group, composition group 1, composition group 2, and recombinant collagen group than in the model group, reaching statistically significant differences (P<0.05). Composition group 1 was significantly higher than composition group 2 and the positive control group (P<0.05).

[0063] For the immobility time in the tail suspension test, the positive control group, composition group 1, composition group 2, and recombinant collagen group were all lower than the model group, and all achieved statistically significant differences (P<0.05). Composition group 1 was lower than the model group and composition group 2, and all achieved statistically significant differences (P<0.05). The recombinant collagen group was also better than composition group 2.

[0064] Regarding the immobility time in the forced swimming test, the value of the composition 1 group was significantly lower than that of the model group and the positive control group (P<0.05).

[0065] The overall experimental results show that composition 1, composition 2, and recombinant collagen can significantly improve the depressive behavior of mice caused by chronic sleep disturbance. The effect of recombinant collagen fusion factor PHPV1.6 is better than protein FLM1.6.0.

[0066] Table 1. Test results

[0067]

[0068]

[0069] Example 4: Sleep improvement effect test

[0070] Healthy SPF ICR mice aged 7 weeks were randomly divided into two groups, half male and half female, with 20 mice in each group.

[0071] The experimental group was gavaged daily with 0.2 mL of the aqueous dispersion of Composition 1 prepared as described in Example 2 (dosage was 20 mg / kg body weight), while the control group was gavaged daily with an equal volume of water for 30 consecutive days.

[0072] On the 31st day, the sleep status of each mouse was monitored using the SPM animal sleep monitoring system, and the results showed that the number of sleep times in the experimental group within 24 hours was 21.7% more than that in the control group (P<0.01), and the total sleep duration within 24 hours was 65.0% more than that in the control group (P<0.001), which proved that Composition 1 had a significant improvement effect on animal sleep.

[0073] Although the embodiments of the present disclosure have been described above, it is understood that the above embodiments are exemplary and do not constitute limitations to the present disclosure. A person skilled in the art may make changes, modifications and variations to the above embodiments within the scope of the present disclosure.

Claims

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

1.

2. A nucleic acid, characterized in that The nucleic acid encodes the protein according to claim 1.

3. A carrier, characterized in that The vector comprises a nucleotide sequence encoding the protein according to claim 1 and a promoter sequence connected to the nucleotide sequence.

4. A composition, characterized in that The composition comprises the protein of claim 1.

5. The composition according to claim 4, characterized in that The composition comprises a protein with an amino acid sequence as shown in SEQ ID NO: 1, γ-aminobutyric acid, calcium gluconate, zinc gluconate, vitamins, oryzanol, a protein with an amino acid sequence as shown in SEQ ID NO: 4, and cordyceps sinensis powder.

6. The composition according to claim 4, characterized in that The composition comprises 0.8-1.2 parts by weight of a protein whose amino acid sequence is shown in SEQ ID NO: 1, 4-6 parts by weight of γ-aminobutyric acid, 4-6 parts by weight of calcium gluconate, 4-6 parts by weight of zinc gluconate, 0.8-1.2 parts by weight of vitamin B1, 0.8-1.2 parts by weight of vitamin B2, 0.8-1.2 parts by weight of vitamin B6, 0.8-1.2 parts by weight of nicotinic acid, 0.8-1.2 parts by weight of oryzanol, 0.8-1.2 parts by weight of a protein whose amino acid sequence is shown in SEQ ID NO: 4, and 0.8-1.2 parts by weight of cordyceps sinensis powder.

7. The composition according to claim 4, characterized in that The composition comprises 1 part by weight of a protein whose amino acid sequence is shown in SEQ ID NO: 1, 5 parts by weight of γ-aminobutyric acid, 5 parts by weight of calcium gluconate, 5 parts by weight of zinc gluconate, 1 part by weight of vitamin B1, 1 part by weight of vitamin B2, 1 part by weight of vitamin B6, 1 part by weight of niacin, 1 part by weight of oryzanol, 1 part by weight of a protein whose amino acid sequence is shown in SEQ ID NO: 4, and 1 part by weight of cordyceps sinensis powder.

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