CN110128503B - Synthetic polypeptide for resisting Abeta 1-42 protein aggregation, synthetic method and application thereof, and gene for encoding synthetic polypeptide - Google Patents
Synthetic polypeptide for resisting Abeta 1-42 protein aggregation, synthetic method and application thereof, and gene for encoding synthetic polypeptide Download PDFInfo
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- CN110128503B CN110128503B CN201910390488.6A CN201910390488A CN110128503B CN 110128503 B CN110128503 B CN 110128503B CN 201910390488 A CN201910390488 A CN 201910390488A CN 110128503 B CN110128503 B CN 110128503B
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Abstract
The invention discloses a synthetic polypeptide for resisting Abeta 1-42 protein aggregation, a synthetic method and application thereof, and a gene for coding the synthetic polypeptide. The synthetic polypeptide provided by the invention has the effects of resisting the Abeta 1-42 protein aggregation activity and relieving cognitive dysfunction, and can effectively prevent or treat Alzheimer Disease (AD) and other neurodegenerative diseases. Therefore, the synthetic polypeptide provided by the invention can be widely applied to research and development of foods or medicines for resisting A beta 1-42 protein aggregation and preventing and treating AD, can improve the medical condition of neurodegenerative diseases to a certain extent, and has great social value and economic benefit.
Description
Technical Field
The invention relates to the technical field of polypeptide, in particular to synthetic polypeptide for resisting Abeta 1-42 protein aggregation, a synthetic method and application thereof, and a gene for encoding the synthetic polypeptide.
Background
Alzheimer's Disease (AD), also known as senile dementia, is a neurodegenerative disease mainly characterized by cognitive dysfunction, and its core symptoms include memory impairment, learning ability decline, self-care ability decline, impaired understanding and judgment of things, behavior mood, etc. The occurrence of AD is a result of combined action of genes, environment and behaviors, with the progress of aging of population and the lack of effective means for predicting and treating AD, the number of AD patients worldwide increases year by year, and the disease becomes the fourth leading cause of death of old people after heart disease, cancer and stroke, so that the development of effective intervention measures and treatment medicines for AD has become a social problem of great concern. The typical pathological features of AD are mainly the following three points: abnormal deposition of amyloid beta (Abeta) and Senile Plaque (SP) formed in brain neurons; secondly, the hyperphosphorylated Tau protein in the nerve cells is abnormally aggregated to form neurofibrillary tangles, and the neuronal cell tangles can cause apoptosis; and thirdly, the neuron loss is accompanied with the proliferation of glial cells. The pathogenesis of AD is very complex, and scientists propose various hypotheses, including a β cascade hypothesis, Tua protein hypothesis, choline function hypothesis, etc., wherein the a β cascade hypothesis is one of the most accepted theories, and at present, the research on AD mainly focuses on the a β cascade hypothesis, which is also the main direction for researching foods and medicines related to AD.
Beta amyloid protein (a β) is a small molecule polypeptide produced by the proteolytic action of transmembrane Amyloid Precursor Protein (APP) in organelles such as endoplasmic reticulum, golgi apparatus, lysosome and the like by beta and gamma secretases. The A beta cascade hypothesis suggests that A beta in human body is in equilibrium state, and when the generated A beta cannot be degraded and cleared in time and is deposited in large quantity outside cells, neurotoxin is generated along with the A beta, thereby triggering AD. Glial cells are the main source of a β in the brain, commonly known as a β, which is often referred to as a β 1-40 or a β 1-42, wherein a β 1-40 tends to form long fibers, mainly playing a role in lengthening fibers, whereas a β 1-42 is more prone to form spherical plaque-like deposits, is more aggregated and less prone to degradation, is more toxic, and is the core component constituting the AD senile plaque. According to the theory, the main targets of preventing, treating and detecting AD are that the generation of A beta 1-42 protein is reduced, the degradation mechanism of A beta 1-42 protein is improved, and the aggregation of A beta 1-42 protein is inhibited.
The main medicines for resisting A beta 1-42 protein aggregation at present comprise homotaurine, melatonin and the like. The homotaurine is a glycosaminoglycan combined with the A beta 1-42 protein monomer and can reduce the polymerization of the A beta 1-42 protein, the second-phase clinical experiment shows that the homotaurine can improve the cognitive function of severe AD patients, the melatonin can antagonize the aggregation of the A beta 1-42 protein, and the research finds that the melatonin can reduce the aggregation of senile plaques in transgenic mice over-expressed by the A beta 1-42 protein. In addition, several bioactive peptides with anti-A β 1-42 protein aggregation are under investigation. The bioactive peptide refers to a peptide compound with good physiological functions for organisms, has wide sources, and comprises natural polypeptide directly separated and extracted from the organisms and synthetic polypeptide synthesized by a chemical or biological mode, wherein the natural polypeptide has the characteristics of long consumed time, high cost, low yield and uncontrollable quality, but the synthetic polypeptide has the characteristics of high purity, low cost, less required time, large-scale batch production and the like, and the polypeptide is synthesized by adopting a solid phase synthesis method at present.
Compared with the prior art, the inhibitor of the polypeptide has good biocompatibility and ecological-like properties, is convenient to synthesize and modify, has good stability, small chance of interaction with an immune system and strong penetrability, and shows good inhibition effect by using the inhibitor based on the peptide, so the peptide and the peptide mimic are promising leads for preventing and treating AD.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a synthetic polypeptide for resisting A beta 1-42 protein aggregation, a synthetic method and application thereof, and a gene for coding the synthetic polypeptide.
The invention aims to provide a synthetic polypeptide for resisting A beta 1-42 protein aggregation.
The invention also aims to provide a method for synthesizing the synthetic polypeptide for resisting the aggregation of the Abeta 1-42 protein.
The invention also aims to provide a gene for coding the synthetic polypeptide resisting the aggregation of the Abeta 1-42 protein.
The invention also aims to provide application of the synthetic polypeptide for resisting the aggregation of the Abeta 1-42 protein.
The purpose of the invention is realized by at least one of the following technical solutions.
The invention provides a synthetic polypeptide for resisting Abeta 1-42 protein aggregation, which is named as LGFH, and has an amino acid sequence of Leu-Gly-Phe-His, as shown in SEQ ID No. 1; wherein Leu is the residue corresponding to the amino acid of leucine, Gly is the residue corresponding to the amino acid of glycine, Phe is the residue corresponding to the amino acid of phenylalanine, and His is the residue corresponding to the amino acid of histidine.
The base sequence of the gene for coding the synthetic polypeptide for resisting the aggregation of the Abeta 1-42 protein is CUCGGCUUCUAC, and the base number is 12bp as shown in SEQ ID No. 2; wherein, CUC is a codon of leucine, GGC is a codon of glycine, UUC is a codon of phenylalanine, and UAC is a codon of histidine.
The invention provides a method for synthesizing the synthetic polypeptide for resisting the aggregation of the Abeta 1-42 protein, which comprises the following steps:
coupling Fmoc protected amino acids and resin one by one according to the amino acid sequence of the synthetic polypeptide for resisting A beta 1-42 protein aggregation from the C end to the N end of the amino acid sequence, then removing the resin and protecting amino acid side chain protecting groups by using a cutting fluid to obtain a crude synthetic polypeptide product, and purifying the crude synthetic polypeptide product to obtain the synthetic polypeptide for resisting A beta 1-42 protein aggregation.
Preferably, the resin is a dichloro resin.
Further, the volume ratio of each component of the cutting fluid is as follows: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%).
Further, the purifying comprises: purifying by high performance liquid chromatography, and performing qualitative analysis by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS) to determine amino acid sequence.
The synthetic polypeptide for resisting A beta 1-42 protein aggregation provided by the invention can be applied to preparation of foods or medicines for resisting A beta 1-42 protein aggregation, and can also be applied to development of foods and medicines for preventing or treating AD.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the synthetic polypeptide for resisting the aggregation of the Abeta 1-42 protein, provided by the invention, has the obvious effect of resisting the aggregation of the Abeta 1-42 protein, can improve the memory and delay the onset of the Alzheimer's disease, and can be widely applied to the research and development of preparing food or medicines for resisting the aggregation of the Abeta 1-42 protein and preventing or treating the Alzheimer's disease. Therefore, the synthetic polypeptide provided by the invention can effectively prevent or treat AD and other neurodegenerative diseases, improves the medical conditions of the neurodegenerative diseases to a certain extent, and has certain social value and economic benefit.
Drawings
FIG. 1a is a high performance liquid chromatogram of the polypeptide LGFH synthesized in example 1;
FIG. 1b is a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS) graph of the polypeptide LGFH synthesized in example 1;
FIG. 2a is a bar graph of the survival rate of MTT cells in example 2 in a negative Control group (Control group), a Model group (Model group), and a synthetic polypeptide at a concentration of 0.05 mM (pre-incubation 24 h), a synthetic polypeptide at a concentration of 0.1 mM (pre-incubation 24 h), a synthetic polypeptide at a concentration of 0.5 mM (pre-incubation 24 h), and a synthetic polypeptide at a concentration of 1 mM (pre-incubation 24 h), wherein ". x" indicates that there is a significant difference between the group and the Control group;
FIG. 2b is a cytogram of the negative control group, the model group, the 0.1 mM polypeptide low dose group and the 0.5 mM polypeptide high dose group at 24h preincubation with the synthetic polypeptides in example 2.
Fig. 2c is a bar graph of the aggregation rates of a β 1-42 proteins in the negative control group, the model group, the polypeptide low dose group having a concentration of 0.1 mM for the synthetic polypeptide (pre-incubation 24 h), and the polypeptide high dose group having a concentration of 0.5 mM for the synthetic polypeptide (pre-incubation 24 h) in example 2, wherein "×" indicates a significant difference between the groups and the control group;
FIG. 3a is a bar graph of the survival rate of MTT cells in example 3 in a negative Control group (Control group), a Model group (Model group), and a synthetic polypeptide added at a concentration of 0.05 mM (48 h pre-incubation), 0.1 mM (48 h pre-incubation), and 0.5 mM (48 h pre-incubation), respectively, to a concentration of 1 mM (48 h pre-incubation), wherein ". about." indicates a significant difference between the group and the Control group;
FIG. 3b is a cytogram of the negative control group, the model group, the polypeptide low dose group with a concentration of 0.1 mM of the synthetic polypeptide (preincubation for 48 h), and the polypeptide high dose group with a concentration of 0.5 mM of the synthetic polypeptide (preincubation for 48 h) in example 3.
Fig. 3c is a bar graph of the aggregation rates of a β 1-42 proteins in the negative control group, the model group, the polypeptide low dose group having a concentration of 0.1 mM for the synthetic polypeptide (preincubation 48 h), and the polypeptide high dose group having a concentration of 0.5 mM for the synthetic polypeptide (preincubation 48 h) in example 3, wherein "×" indicates a significant difference between the groups and the control group.
Detailed Description
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
In a specific embodiment, the synthetic polypeptide resisting Ass 1-42 aggregation provided by the invention is named as LGFH, and the amino acid sequence is as follows: Leu-Gly-Phe-His as shown in sequence table SEQ ID No. 1;
wherein Leu is the residue corresponding to the amino acid of leucine, Gly is the residue corresponding to the amino acid of glycine, Phe is the residue corresponding to the amino acid of phenylalanine, and His is the residue corresponding to the amino acid of histidine.
The synthetic polypeptide for resisting A beta 1-42 protein aggregation can be synthesized by a polypeptide solid phase synthesis method or a gene engineering technology;
wherein, when the polypeptide is synthesized by a polypeptide solid phase synthesis method, a standard Fmoc scheme is adopted, and dichloro resin is selected; fmoc is adopted to protect the N end of amino acid, and each protected amino acid is Fmoc-L-Leu-OH, Fmoc-Gly-OH, Fmoc-L-Phe-OH and Fmoc-L-His-OH. The protected amino acids and the resin are coupled one by using coupling reagents and activating reagents which are conventional in the field of solid phase synthesis, including 4-Dimethylaminopyridine (DMAP) and Dicyclohexylcarbodiimide (DCC) for completing the coupling of the first protected amino acid and the resin, and 1-hydroxybenzotriazole (HOBt) for performing the coupling of the remaining protected amino acids. According to the sequence of the amino acids from the C end to the N end of the polypeptide, coupling the protected amino acids with the resin one by one, then removing the resin and protecting the side chain protecting groups of the protected amino acids from the lysate to obtain a crude product of the synthetic polypeptide, and purifying the crude product of the synthetic polypeptide to obtain the synthetic polypeptide resisting the aggregation of the Abeta 1-42 protein.
In a specific embodiment, the invention is based on an Abeta cascade hypothesis, and an in vitro research on AD senile plaque aggregation is carried out on the prevention of synthetic polypeptide LGFH by adopting an E22G-Abeta 42-mCherry HEK-293 transgenic cell model.
In vitro experiments were carried out using tetracycline to induce E22G-mcherry Hek-293 cell line. The tetracycline can induce the A beta protein to be aggregated in cells to generate toxicity, and can simulate the pathological development process of senile plaques of neuron cells in AD patients. Tetracycline induces the A beta protein to form red fluorescent protein in cells and emit red fluorescence, and a CytoFlexS flow cytometer is adopted to carry out positioning and quantitative detection on the mcherry red fluorescence.
Example 1
Solid phase synthesis method for synthesizing polypeptide LGFH
1. Synthetic route process flow
Using dichloro resin as a carrier, firstly swelling the resin, then reacting C-terminal carboxyl of a first amino acid with active site chlorine on the resin, after the first amino acid is connected on the resin, carrying out dehydration condensation to connect a second amino acid, and removing Fmoc protection after the condensation is finished. Repeating the operation according to the amino acid sequence shown in SEQ ID No. 1, sequentially connecting the rest amino acids from the C end to the N end, and finally cutting the polypeptide from the resin by using a cutting reagent, wherein the volume ratio of each component in the cutting reagent is TFA (94.5%), water (2%), EDT (2.5%), and TIS (1%).
2. Synthesis process
Using dichloro resin as a carrier, firstly swelling the dichloro resin, weighing 1.28 g of the dichloro resin, putting the dichloro resin into a reaction column, then adding 20 mL of DCM reagent into the reaction column, oscillating for 30 min, and activating for later use. The first amino acid was ligated, DCM solvent was removed by suction through a sand core, Fmoc-L-His-OH, 1.05 times the molar amount of the dichloro resin, DIEA (diisopropylethylamine), 10 times the molar amount of the dichloro resin, finally 10 mL of DMF was added for dissolution, and shaking was carried out for 1 h. After the reaction was complete, the reaction was washed 6 times with DMF and DCM alternately. 20 mL of a DMF solution containing 20% by volume of piperidine was added, and the DMF solution was removed after 5 min. Then 20 mL of DMF solution containing 20% by volume of piperidine was added, and the mixture was shaken for 15 min for deprotection. Then, removing the piperidine solution, taking 15 particles of resin (dichloro resin), washing with ethanol for three times, adding ninhydrin, pyridine and phenol one drop by drop, heating at 105-110 ℃ for 5 min, turning dark blue to be a positive reaction, continuing to prepare the next amino acid, and if the color is not changed, turning negative to be deprotected again. Next, the first washing was performed, and the washing was performed twice with 15 mL of DMF (N, N-dimethylformamide), 15 mL of methanol, and 15 mL of DMF in this order. Fmoc-L-Phe-OH in an amount of 3 times the molar amount of the resin (dichloro resin) and HBTU in an amount of 3 times the molar amount of the resin were added, and the mixture was dissolved in 10 mL of DMF, and DIEA in an amount of 10 times the molar amount of the resin was added and reacted for 30 min to effect condensation. A second wash was then performed, using 15 ml DMF, 15 ml methanol and 15 ml DMF in sequence, twice. Removing the solvent, taking 15 particles of dichloro resin, washing with ethanol for three times, adding 200mL of ninhydrin, pyridine and phenol respectively, heating at 105-110 ℃ for 5 min, wherein the colorless state is a positive reaction, and the condensation is needed again if the color is blue. Repeating the operation according to the method, and sequentially connecting the rest amino acids to complete the extension of the peptide chain. After the last amino acid has been grafted, the synthesis of the whole peptide is completed, washing with DMF for 3 times, DCM for 3 times, methanol for 3 times, and finally draining the peptide resin to complete the final contraction phase.
3. Deprotection of amino acid side chains and cleavage of resins
Preparing 15 mL of cutting fluid, wherein the volume percentage of each component of the cutting fluid is as follows: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%). The resin (dichloro resin) was charged into a flask and shaken at a constant temperature (30 ℃ C.) for 2 hours. The lysate was blown dry with nitrogen, then poured into a centrifuge tube and 40 mL of diethyl ether was poured. Sealing and placing in a centrifuge for 5 min at 3000rpm, and pouring off the supernatant to leave a white precipitate at the bottom. Washing with ether for 6 times, and volatilizing at normal temperature to obtain crude peptide.
4. HPLC purification
The crude peptide is put into a vessel, dissolved completely by 30-50 mL of acetonitrile water solution with volume fraction of 50%, and subjected to ultrasonic treatment for 2 min. The resulting solution was filtered through a 0.45 μm filter. 3 μ l of the lysate was analyzed by analytical grade HPLC for crude peptide for subsequent preparation. The mobile phase is water and acetonitrile, the time is 30 min, gradient elution is carried out, HPLC is firstly balanced for 5 min by using an initial gradient, then sample injection is carried out, and the volume ratio of the initial gradient is as follows: 95% of water, 5% of acetonitrile and the volume ratio of the finished gradient is as follows: 5% of water and 95% of acetonitrile. And preparing a sample injection preparation for the dissolved sample. Preparative HPLC equilibrated for 10 min with an initial gradient: water 95%, acetonitrile 5%, end gradient: 25% of water, 75% of acetonitrile and 40 min of gradient time. The sample from the detector is collected. The above processes are all completed in a SYMPHONY type 12-channel polypeptide synthesizer, the synthesized polypeptide (the synthetic polypeptide resisting Abeta 1-42 protein aggregation) is purified by a SHIMADZU high performance liquid chromatograph, the purity reaches more than 99 percent, and the amino acid sequence is determined by adopting a liquid chromatography-mass spectrometry/mass spectrometry combined technology (LC-MS).
The high performance liquid chromatogram and the liquid chromatography-mass spectrum/mass spectrum (LC-MS) of the synthesized polypeptide are respectively shown in figure 1a and figure 1b, and the analysis of figure 1a and figure 1b shows that the primary amino acid sequence of the synthesized polypeptide is Leu-Gly-Phe-His, thus obtaining the target polypeptide, and synthesizing to obtain the synthetic polypeptide for resisting the aggregation of Abeta 1-42 protein.
Example 2
Synthetic polypeptide LGFH (synthetic polypeptide resisting A beta 1-42 protein aggregation) preincubation for 24h in vitro activity experiment resisting A beta 1-42 protein aggregation
1. Experimental methods
Preparation of a culture medium: 40 mL of complete medium was prepared from 35 mL of high-glucose medium (DMEM), 4 mL of Fetal Bovine Serum (FBS), 1 mL of L-glutamine, 40. mu.L of Hygromycin B and 20. mu.L of Blasticidin S antibiotic.
Preparation of synthetic polypeptide (LGFH) solution: 4.543 mg of polypeptide LGFH was weighed out and dissolved in 10 mL of the complete medium, and after passing through a 0.22 μm frit, the stock solution was 1 mM, and the stock solution was diluted with the complete medium to the concentration required for the above experiment.
Preparing a 2 mg/mL tetracycline solution: 10 mg of tetracycline was weighed, prepared with 5 mL of 1 Í PBS buffer, filtered through a 0.22 μm filter, and stored at-20 ℃ in the dark for use.
Cultures and experiments were performed using E22G-mcherry Hek-293 cells. Grouping experiments: negative Control group (Control group, E22G-mcherry Hek-293 cells, without tetracycline induction); model group (Model group, E22G-mcherry Hek-293 cells, induced with tetracycline); polypeptide LGFH low dose group (0.1 mM) and polypeptide LGFH high dose group (0.5 mM), each group set three replicates.
Using 6-well plate for cell plating, the number of cells in each well is 20000, after cells grow 24h adherent, adding the complete culture medium and the synthetic polypeptide solution (LGFH polypeptide solution) according to experimental groups. After culturing for 24h, changing the culture solution of each group, adding the complete culture medium and the synthetic polypeptide solution (LGFH polypeptide solution) according to experimental groups, wherein tetracycline is required to be added in a model control group, a polypeptide LGFH low dose group (0.1 mM) and a polypeptide LGFH high dose group (0.5 mM), and the mass fraction of the tetracycline in the complete culture medium is 10 mu g/mL, and continuing to induce and culture for 72 h. Detecting mcherry red fluorescence by adopting a CytoFlexS flow cytometer, and adopting an ECD channel under a 561 exciter. Wherein, the calculation of the A beta aggregation rate is as follows:
Α β aggregation rate = treatment group mean fluorescence intensity/model group intensity 100%.
Example 3
Synthetic polypeptide LGFH (synthetic polypeptide resisting A beta 1-42 protein aggregation) preincubation for 48 h in vitro activity experiment resisting A beta 1-42 protein aggregation
1. Experimental methods
Preparation of a culture medium: 40 mL of complete medium was prepared from 35 mL of high-glucose medium (DMEM), 4 mL of Fetal Bovine Serum (FBS), 1 mL of L-glutamine, 40. mu.L of Hygromycin B and 20. mu.L of Blasticidin S antibiotic.
Preparation of synthetic polypeptide (LGFH) solution: 4.543 mg of polypeptide LGFH was weighed out and dissolved in 10 mL of the complete medium, and after passing through a 0.22 μm frit, the stock solution was 1 mM, and the stock solution was diluted with the complete medium to the concentration required for the above experiment.
Preparing a 2 mg/mL tetracycline solution: 10 mg of tetracycline was weighed, prepared with 5 mL of 1 Í PBS buffer, filtered through a 0.22 μm filter, and stored at-20 ℃ in the dark for use.
Cultures and experiments were performed using E22G-mcherry Hek-293 cells. Grouping experiments: negative Control group (Control group, E22G-mcherry Hek-293 cells, without tetracycline induction); model group (Model group, E22G-mcherry Hek-293 cells, induced with tetracycline); polypeptide LGFH low dose group (0.1 mM) and polypeptide LGFH high dose group (0.5 mM), each group was three replicates.
Using 6-well plate for cell plating, the number of cells in each well is 20000, after cells grow 24h adherent, adding the complete culture medium and the synthetic polypeptide solution (LGFH polypeptide solution) according to experimental groups. After culturing for 48 h, changing the culture solution of each group, adding the complete culture medium and the synthetic polypeptide solution (LGFH polypeptide solution) according to experimental groups, wherein tetracycline needs to be added additionally in the model control group, the polypeptide LGFH low dose group (0.1 mM) and the polypeptide LGFH high dose group (0.5 mM), and the mass fraction of the tetracycline in the complete culture medium is 10 mu g/mL, and continuing to induce and culture for 72 h. Detecting mcherry red fluorescence by adopting a CytoFlexS flow cytometer, and adopting an ECD channel under a 561 exciter. Wherein, the calculation of the a β aggregation rate is as follows:
Α β aggregation = treatment group mean fluorescence intensity/model group intensity 100%.
2. Results of the experiment
The negative Control group and the blank Control group (Control group) were cells induced without adding tetracycline, the Model group (Model group) was cells induced with adding tetracycline, and the LGFH was an experimental group added with synthetic polypeptide LGFH at different concentrations. The MTT cell viability map of the negative control group and the synthetic polypeptide LGFH pre-incubated for 24h at different concentrations is shown in FIG. 2 a; the partial flow chart and the A beta 1-42 protein aggregation rate bar chart of the negative control group, the model control group, the 0.1 mM polypeptide low dose group and the 0.5 mM polypeptide high dose group which are pre-incubated for 24h are respectively shown in the figures 2b and 2 c;
as shown in example 2, when the polypeptide was preincubated for 24 hours, the survival rate of the cells was concentration-dependent, and the inhibition phenomenon was observed at a high concentration, as shown in FIG. 2 a. The polypeptide has the cell proliferation promoting phenomenon at the concentration of 0.1 mM but has no significant difference with a control group, the cell survival rate is between 80% and 100% at the concentration of 0.5 mM, namely, no cytotoxicity exists, so that two concentrations of 0.1 and 0.5 mM can be selected for subsequent experiments; as can be seen from the partial local flow chart of fig. 2b, the mCherry fluorescence intensity of the model control group is higher than that of the negative control group, and the mCherry fluorescence intensity of the polypeptide administration group is reduced compared with that of the model control group. The mCherry mean fluorescence intensity is used to show the aggregation rate of the Abeta 1-42 protein, and as can be seen from the protein aggregation bar chart of FIG. 2c A beta 1-42, the aggregation rate of the polypeptide Abeta 1-42 protein in the model control group is significantly increased compared with that in the negative control group, which indicates that the modeling is successful. Compared with a model control group, the A beta 1-42 protein aggregation rate of a polypeptide administration group with high (0.5 mM) and low (0.1 mM) doses is remarkably reduced, wherein the A beta 1-42 protein aggregation rate of the polypeptide low dose group is reduced more, which shows that the polypeptide LGFH can reduce the A beta 1-42 protein aggregation rate, and the anti-A beta 1-42 protein aggregation effect of the low dose group is better than that of the high dose group.
The MTT cell viability map of the negative control group and the synthetic polypeptide LGFH pre-incubated for 48 h at different concentrations is shown in FIG. 3 a; the partial flow chart and the bar chart of the A beta 1-42 protein aggregation rate of the negative control group, the model control group, the 0.1 mM polypeptide low dose group and the 0.5 mM polypeptide high dose group which are pre-incubated for 48 h are respectively shown in the figures 3b and 3 c.
As shown in example 3, when the polypeptide was preincubated for 48 h, the cell survival rate was concentration-dependent and high-concentration inhibition was observed in FIG. 3 a. The polypeptide has the effect of promoting cell proliferation when the concentration is 0.1 mM but has no significant difference with a control group, the cell survival rate is between 80 and 100 percent when the concentration is 0.5 mM, and no cytotoxicity exists, so that two concentrations of 0.1 and 0.5 mM can be selected for subsequent experiments; as can be seen from the partial local flow chart of fig. 3b, the mCherry fluorescence intensity of the model control group is higher than that of the negative control group, and the mCherry fluorescence intensity of the polypeptide administration group is reduced compared with that of the model control group. The mCherry mean fluorescence intensity is adopted to show the aggregation rate of the Abeta 1-42 protein, and as can be seen from a protein aggregation diagram of 3c A beta 1-42, compared with a negative control group, the aggregation rate of the polypeptide Abeta 1-42 protein in the model control group is obviously increased, which indicates that the modeling is successful. Compared with a model control group, the A beta 1-42 protein aggregation rates of the polypeptide administration groups with high (0.5 mM) and low (0.1 mM) doses are remarkably reduced, wherein the A beta 1-42 protein aggregation rate of the polypeptide low dose group is reduced more, which indicates that the polypeptide LGFH can reduce the A beta 1-42 protein aggregation rate, and the anti-A beta 1-42 protein aggregation effect of the low dose group is better than that of the polypeptide high dose group.
The results show that the polypeptide preincubation time with the same concentration, no matter 24h or 48 h, has the effect of resisting the aggregation of the Abeta 1-42 protein; when the polypeptide preincubation time is the same, the polypeptide concentration of high and low doses has the effect of resisting A beta 1-42 protein aggregation, and the anti-A beta 1-42 protein aggregation effect of the 0.1 mM polypeptide low dose group is better than that of the polypeptide high dose group. Therefore, 0.1 mM synthetic polypeptide LGFH pre-incubation for 24h and 48 h has better effects of improving memory and inhibiting the disease process of AD, can be applied to preparing food or medicine for resisting A beta 1-42 protein aggregation and preventing and treating AD, and can effectively prevent and treat neurodegenerative diseases including AD diseases.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Sequence listing
<110> university of southern China's science
<120> synthetic polypeptide for resisting Abeta 1-42 protein aggregation, synthetic method and application thereof, and gene for coding synthetic polypeptide
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> Artificial Synthesis (Artificial sequence)
<400> 1
Leu Gly Phe His
1
<210> 2
<211> 12
<212> DNA/RNA
<213> Artificial Synthesis (Artificial sequence)
<400> 2
Claims (7)
1. A synthetic polypeptide for resisting Abeta 1-42 protein aggregation is characterized in that the name is LGFH, the amino acid sequence is Leu-Gly-Phe-His, and the amino acid sequence is shown as SEQ ID No. 1; wherein Leu is the residue corresponding to the amino acid of leucine, Gly is the residue corresponding to the amino acid of glycine, Phe is the residue corresponding to the amino acid of phenylalanine, and His is the residue corresponding to the amino acid of histidine.
2. A gene encoding the synthetic polypeptide of claim 1, wherein the synthetic polypeptide has the base sequence of CUCGGCUUCUAC, and has the base number of 12bp as shown in SEQ ID No. 2; wherein, CUC is a codon of leucine, GGC is a codon of glycine, UUC is a codon of phenylalanine, and UAC is a codon of histidine.
3. A method for synthesizing a synthetic polypeptide according to claim 1 which is resistant to aggregation of a β 1-42 protein, comprising the steps of:
coupling Fmoc protected amino acids from the first amino acid at the C end and resin one by one according to the amino acid sequence of the synthetic polypeptide for resisting A beta 1-42 protein aggregation from the C end to the N end of the amino acid sequence, then performing dehydration condensation between the amino acids one by using a solid-phase synthesis method to form a peptide chain, removing the resin and a protecting group by using a cutting fluid to obtain a crude synthetic polypeptide, and purifying the crude synthetic polypeptide to obtain the synthetic polypeptide for resisting A beta 1-42 protein aggregation.
4. The method of synthesis according to claim 3, characterized in that the resin is a dichloro resin.
5. The method of claim 3, wherein the cleavage solution comprises, by volume, 94.5% TFA, 2% water, 2.5% EDT, and 1% TIS.
6. The method of claim 3, wherein the purification is performed by HPLC and qualitative analysis using LC-MS/MS technology to determine the amino acid sequence.
7. The use of a synthetic polypeptide according to claim 1, which is resistant to a β 1-42 protein aggregation, for the preparation of a medicament for the prevention or treatment of alzheimer's disease.
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