CN110551195B - Alpha-synuclein aggregation-induced emission system and construction and application thereof - Google Patents

Abstract

An α-synuclein aggregation-induced luminescence system and its construction and application. The invention belongs to the fields of biotechnology and chemical engineering, and in particular relates to an αSN aggregation-induced luminescence fusion and a construction method and application thereof. The αSN aggregation-induced luminescence fusion is specifically EPB-αSN4F or EPB-αSN39Y, which is to replace the phenylalanine at the 4th position or the tyrosine at the 39th position in the αSN amino acid sequence with p-azidophenylalanine. pAZF, obtain αSN4F or αSN39Y mutant protein, and then carry out bioorthogonal ligation reaction of AIE molecule EPB with mutant protein αSN4F or αSN39Y to obtain EPB-αSN4F or EPB-αSN39Y, which can be used for the screening of aggregation inhibitors.

Description

An α-synuclein aggregation-induced luminescence system and its construction and application

Technical field:

The invention belongs to the fields of biotechnology and chemical engineering, in particular to the construction of an α-synuclein aggregation-induced luminescence system, and the system is used in the screening of α-synuclein aggregation inhibitors. Specifically, the expression and purification of α-synuclein containing unnatural amino acid in Escherichia coli, the traceless addition of aggregation-induced luminescent probes, and the screening of aggregation inhibitors.

Background technique:

The aggregation and misfolding of amyloid in cells and tissues to form toxic intermediates and amyloid fibrils may lead to various biological dysfunctions. These aggregated intermediates and fibers are presumed to be associated with some neurodegenerative diseases and other disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), diabetes type II (diabetes type II) )Wait. Numerous studies have demonstrated that the more toxic fractions are oligomers or fibrillar intermediates, and mature fibers may also play an important role. The development of effective amyloid aggregation inhibitors has become one of the effective means in the field of drug development for the treatment of these diseases. Therefore, sensitive and convincing methods for the detection of amyloid fibrils or intermediates and detailed understanding of the fibril formation mechanism at the molecular level are required. This knowledge can help rationalize the design of therapeutic regimens to prevent and slow down the damage to the body caused by toxic amyloid fragments. In recent years, some breakthroughs have been made in the research on the structure, morphology, and full length of amyloid fibrils. However, the molecular-level aggregation mechanism of amyloid misfolding remains unexplored.

At present, the screening methods of amyloid aggregation inhibitors mainly include the following three aspects: (1) In vitro dye experiment screening. Amyloid dyes such as Thioflavin T (ThT) and Congo red (CR) are sensitive in a hydrophobic environment and fluoresce when bound to the β-sheet structure of amyloid fibrils, and are therefore widely used. Aggregated protein research and high-throughput screening and identification of aggregation inhibitors. However, the application of these dyes has many limitations. For example, the morphology of early aggregates cannot be detected with these dyes, and they cannot be used to accurately explore small molecules because small-molecule drugs may compete with these dyes for binding to amyloid fibrils. Morphological changes in fibers induced by molecular drugs. In addition, the properties of the dye may be affected by other components in the buffer solution, so it is prone to some false positives, etc. (2) Fluorescent protein marker screening. To visualize the aggregation process, green fluorescent protein GFP was expressed as a fusion protein with β-amyloid 42 (Aβ42) and used to screen for Aβ42 aggregation inhibitors. However, due to the large molecular weight of GFP, the aggregation and folding of only 4.5kDa Aβ42 protein is difficult to cause the misfolding of the entire fusion protein. Therefore, the aggregation and folding process of GFP-Aβ42 fusion protein may not be completely the aggregation process of Aβ42. In addition, the background fluorescence of dyes and fluorescent proteins may greatly affect the detection results. (3) Molecular design and virtual screening. With the rapid development of molecular simulation technology in recent years, molecular simulation techniques, such as molecular dynamics simulation, molecular docking and pharmacophore modeling, have been widely used to analyze the mechanism of amyloid aggregation and misfolding and its inhibition, computational Types of binding forces between inhibitors and amyloid, determination of inhibitor sites, and screening and design of aggregation inhibitors. However, due to the unstable conformation of amyloid aggregates, especially the three-dimensional structures of some of the most toxic oligomers have not yet been resolved, the application of molecular modeling technology in the development of amyloid aggregation inhibitors has been severely restricted.

Aggregation-Induced Emission (AIE) probes have the characteristics of not fluorescing in the free state, but can fluoresce strongly when aggregates are formed or when their conformation is restricted, so they can be used as the best choice for analyzing environmental and conformational changes. biological sensor. The principle of aggregation-induced luminescence may be that the intramolecular rotation of the probe is restricted by the environment, resulting in a local excited state, which produces an abnormal photophysical effect. This conformation-switch-dependent luminescence phenomenon is not disturbed by background fluorescence, making it applicable to the biodynamic studies of amyloid. In recent years, several AIE molecules have been reported to detect and identify amyloid fibrils and explore the relationship between proteins and proteins, including TPE, TPE-TPP, BSPOTPE, EPB, etc. However, imperfectly, these AIE molecules have shortcomings such as weak sensitivity and specificity in the process of application.

In order to solve the problem in the current screening of amyloid aggregation inhibitors, the present invention combines the aggregation-induced luminescence technology to introduce unnatural amino acids (Unnatural Amino Acid, UAA) into α-synuclein (α-synuclein, αSN), and then The azide group on its side chain is coupled with the AIE molecule through a bioorthogonal reaction, and the labeling of the AIE molecule is carried out on the αSN, thereby improving the specificity and sensitivity of AIE for detecting protein conformational transition, and obtaining AIE-mutαSN Biosensor and application in the screening of amyloid aggregation inhibitors.

Invention content:

In order to achieve the above purpose, the present invention combines the biological expression of unnatural amino acids and the bioorthogonal reaction technology to construct an αSN aggregation-induced luminescence system, which is applied to the screening of αSN aggregation inhibitors.

One of the technical solutions provided by the present invention is an αSN aggregation-induced luminescence fusion. The αSN aggregation-induced luminescence fusion is specifically EPB-αSN4F or EPB-αSN39Y, which is a combination of phenylalanine at position 4 in the αSN amino acid sequence. Acid or tyrosine at position 39 was replaced by p-Azido-L-phenylalanine (pAZF) to obtain αSN4F or αSN39Y mutant protein, and then AIE molecule EPB was combined with mutant protein αSN4F or αSN39Y was subjected to a bioorthogonal ligation reaction to obtain EPB-αSN4F or EPB-αSN39Y.

The amino acid sequence of αSN is shown in SEQ ID NO. 2 of the sequence table.

The chemical formula of the AIE molecule EPB is: C ₃₈ H ₄₄ N ₂ O ₂ Br ₂ , and the structural formula is shown in Figure 1-a.

The present invention also provides a construction method of EPB-αSN4F or EPB-αSN39Y, which is specifically as follows:

(1) Obtain a protein expression gene by replacing pAZF with phenylalanine at position 4 or tyrosine at position 39 in αSN3 by gene synthesis or PCR, and obtain αSN4F or αSN39Y mutant by expression in E. coli protein;

(2) The copper-catalyzed azide-terminal alkyne cycloaddition reaction (CuAAC) was used to bioorthogonally link EPB with UAA mutant protein αSN4F or αSN39Y to obtain EPB-αSN4F or EPB-αSN39Y.

The present invention also provides the application of EPB-αSN4F or EPB-αSN39Y in the screening of αSN aggregation inhibitors. The fluorescence intensity was detected within the range of -600 nm, and if the fluorescence intensity was weaker than that of the EPB-αSN4F or EPB-αSN39Y system without the potential inhibitor, the potential inhibitor was determined to be an αSN aggregation inhibitor.

When there is an inhibitor in EPB-αSN4F or EPB-αSN39Y solution, it can inhibit the aggregation of αSN4F or αSN39Y, resulting in no or weak luminescence of the screening system. The screening system can emit strong fluorescence;

Preferably, the concentration of EPB-αSN4F or EPB-αSN39Y is 20 μM, the excitation wavelength is 350 nm, and the fluorescence intensity is detected within the range of emission wavelength 380 nm-600 nm, preferably emission wavelength 490 nm.

Beneficial effects:

EPB cannot generate fluorescence with the aggregation of αSN, that is, EPB cannot be used alone to study the aggregation characteristics of αSN; αSN mutated by pAzF cannot make EPB aggregate to generate fluorescence during the aggregation process; EPB-αSN4F or EPB-αSN4F constructed in the present invention or EPB-αSN39Y can induce the system to emit strong fluorescence when forming aggregates, thus providing a new method for the screening of αSN aggregation inhibitors.

Description of drawings:

Fig.1 EPB molecular structure and verification of aggregation-induced luminescence properties

Among them, a. EPB molecular structure; b. EPB fluorescence value changes in different concentrations of glycerol;

Figure 2 The flow chart of the construction of the αSN aggregation-induced luminescence system;

Figure 3. Protein expression, purification and aggregation properties of αSN and six UAA mutants

Among them, a. structure and replacement principle of unnatural amino acid pAzF; b. pAzF replacement site in αSN protein; c. schematic diagram of pET22b-αSN/mutαSN expression vector; d. SDS-PAGE gel electrophoresis to detect αSN and its six pAzF mutations somatic protein;

Figure 4 Identification of αSN and its six pAzF mutant proteins by MALDI TOF mass spectrometry

Wherein, a-g are respectively: αSN, αSN4F, αSN39Y, αSN94F, αSN125Y, αSN133Y, αSN136Y;

Figure 5. ThT fluorescent staining to detect the aggregation properties of αSN and its six pAzF mutants;

Figure 6 Construction and verification of EPB-αSN4F/39Y system

Among them, a. Using CuAAC to connect EPB to αSN4F/39Y to construct EPB-αSN4F/39Y; b, c. UV-visible spectroscopy full wavelength scan to verify whether EPB-αSN4F and EPB-αSN39Y were successfully constructed, the scanning wavelength range is 200 -600nm;

Figure 7 Application of EPB-αSN4F/39Y aggregation-induced luminescence system wherein, (a) EPB-αSN4F; (b) EPB-αSN39Y system;

Figure 8 Aggregation characteristics of different luminescent systems

Among them, a is the αSN4F-related system; b is the αSN39Y-related system.

Detailed ways:

In order to make the purpose, technical solutions and advantages of the present patent more clear, the present patent will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present patent, but not to limit the present invention.

In the present invention, restriction endonuclease and DNA polymerase were purchased from Dalian Bao Bioengineering Co., Ltd., a small amount of plasmid extraction kit, DNA gel recovery kit, and DNA purification recovery kit were purchased from Omega bio-tech, and Ni ⁺ resin was purchased from Omega bio-tech. The unnatural amino acid pAzF was purchased from Sigma Company, and the unnatural amino acid pAzF was purchased from MCE Company, and other reagents were purchased from Shanghai Yuanye Biotechnology Co., Ltd. if the source was not specified. Gene and primer synthesis were obtained from Suzhou Jinweizhi Biotechnology Co., Ltd. The aggregation-induced luminescent molecule EPB was synthesized by entrusting a chemical synthesis formula. If the experimental operation is not described in detail, the operation is carried out according to the laboratory manual - such as "Molecular Cloning".

Tetrastyrene and its derivatives are the most deeply studied AIE molecules due to their simple synthesis and easy modification. The EPB molecule is a derivative of tetraphenylene.

The AIE molecule selected in the present invention is EPB, the full name is 2,20-(((2-(4-ethynylphenyl)-2-phenylethene-1,1-diyl)bis(4,1-phenylene))bis(oxy))bis -(N,N,N-trimethylethanaminium)bromide, the molecule is a derivative of tetraphenylene with good aggregation-induced luminescence properties, which have been published in Hu, R.; Yap, H.K.; Fung, Y.H.; Wang, Y .;Cheong,W.L.;So,L.Y.;Tsang,C.S.;Lee,L.Y.;Lo,W.K.;Yuan,J.;Sun,N.;Leung,Y.C.;Yang,G.;Wong,K.Y.,'Light up'protein -protein interaction through bioorthogonal incorporation of a turn-onfluorescent probe into beta-lactamase. Mol. Biosyst. 2016, 12(12), 3544-3549. Those skilled in the art can synthesize EPB according to the above documents or relevant information such as structural formula and chemical formula.

The unnatural amino acid selected in the present invention is a phenylalanine structural analog containing an azide group, p-Azido-L-phenylalanine (pAZF). In order to reduce the effect of the substitution of UAA on the aggregation properties of the protein itself, according to the principle of structural similarity, phenylalanine (positions 4 and 94) and tyrosine (positions 39, 125, 133 and 136) in αSN were selected as Replacement site for pAzF. Six kinds of pAzF mutant αSN isoform proteins were obtained by expression and purification in E. coli. The aggregation properties of the mutants were analyzed by ThT fluorescence staining, and the results showed that the aggregation of αSN4F and αSN39Y was less affected by the introduction of pAzF, so αSN4F or αSN39Y mutant proteins were selected for subsequent experiments.

The present invention will be further explained and illustrated by specific embodiments below. The construction flow of the αSN aggregation-induced luminescence screening system is shown in Figure 2.

Example 1: Selection of aggregation-induced luminescent molecules and identification of luminescent properties

The aggregation-induced luminescent molecule EPB is a derivative of tetraphenylene, and its structural formula is shown in Figure 1-a. In order to verify the aggregation-inducing properties of the AIE molecule, the same concentration of EPB (2 μM) was used to detect the change of its fluorescence value in different concentrations of glycerol. as PBS, pH 7.4. After thorough mixing, the fluorescence was detected under the conditions of excitation wavelength of 350 nm and emission wavelength of 460 nm.

The experimental results are shown in Figure 1-b, the fluorescence intensity of EPB solution gradually increased with the increase of glycerol concentration, which proved that it indeed has the characteristics of aggregation-induced luminescence.

Example 2: Expression and purification of αSN isoforms containing unnatural amino acid pAzF

(1) Select phenylalanine (positions 4 and 94) and tyrosine (positions 39, 125, 133 and 136) in αSN as the replacement sites of pAzF, and obtain the 4th or 94th by gene synthesis or PCR The coding genes of αSN4F, αSN39Y, αSN94F, αSN125Y, αSN133Y, αSN136Y in which the phenylalanine, 9, 125, 133 or 136 tyrosine of pAzF are replaced (replacement principle and replacement site are shown in Figure 3-a, 3 -b);

The gene encoding the αSN protein was synthesized by Jinweizhi Company after codon optimization. The gene sequence is shown in SEQ ID No. 1. The αSN125Y amber mutant gene was also synthesized by the gene company, and the other five amber mutant genes were synthesized by using the wild-type gene as The template is obtained by PCR or overlapping PCR, and the primers used are shown in SEQ ID No.3-No.11.

The primers for cloning αSN4F gene were NdeI-4αSN-F/XhoI-αSN-R.

When cloning the αSN39Y gene, first use the primer NdeI-αSN-F/39TAG-R to clone the left arm fragment, use the primer 39TAG-F/XhoI-αSN-R to clone the right arm fragment, and then use the left and right arm fragment mixture as a template, use the primer NdeI - αSN-F/XhoI-αSN-R clone αSN39Y gene.

When cloning the αSN94F gene, first use the primer NdeI-αSN-F/94TAG-R to clone the left arm fragment, use the primer 94TAG-F/XhoI-αSN-R to clone the right arm fragment, and then use the left and right arm fragment mixture as a template, use the primer NdeI - αSN-F/XhoI-αSN-R clone αSN94Y gene.

The primer of the cloned αSN133Y gene was NdeI-αSN-F/133TAG-R.

The primer of the cloned αSN136Y gene was NdeI-αSN-F/136TAG-R.

NdeI-αSN-F: GGAATTCCATATG GATGTGTTTATGAAAGGCCT (SEQ ID No. 3);

XhoI-αSN-R: CCGCTCGAG GGCTTCCGGTTCATAATCCT (SEQ ID No. 4);

NdeI-4αSN-F: GGAATTCCATATGGATGTGTAGATGAAAGGCCT (SEQ ID No. 5);

39TAG-R: TTGCTACCCACCTACAGCACGCCCT (SEQ ID No. 6);

39TAG-F: AGGGCGTGCTGTAGGTGGGTAGCAA (SEQ ID No. 7);

94TAG-R: CTTTCTTCACCTAACCGGTGGCG (SEQ ID No. 8);

94TAG-F: CGCCACCGGTTAGGTGAAGAAAG (SEQ ID No. 9);

133TAG-R: CCGCTCGAGGGCTTCCGGTTCATAATCCTGCTAGCCAA (SEQ ID No. 10);

136TAG-R: CCGCTCGAGGGCTTCCGGTTCCTAATCCTGGTAGCC (SEQ ID No. 11).

(2) In order to facilitate the screening and purification of the UAA mutant protein in the later stage, the pET22b plasmid was selected as the expression vector.

(3) An expression vector for expressing wild-type αSN and 6 mutant proteins in E. coli was constructed using the conventional chemical transformation method of E. coli. The schematic diagram of the expression vector is shown in Figure 3-c.

For the wild-type αSN protein, the pET22b-αSN expression vector was transformed into the E. coli BL21 expression host to construct the corresponding recombinant strain BL21-αSN. Pick a single colony of the constructed BL21-αSN engineering bacteria into 5mL LB medium for overnight culture at 37°C, transfer to fresh LB medium at 1% inoculum, and culture at 37°C until the OD600 is 0.6-0.8, and add a final concentration of 0.5 Induction by mMIPTG, the induction temperature was 16 °C, and the induction time was 16-18 h. Finally, the cells were collected by centrifugation at 6000 rpm for 10 min. The bacteria obtained above were resuspended with a buffer lysisbuffer (20mM Tris-HCl, pH 7.4, 200mMNaCl, 1mM EDTA, 1mM DTT), added with a final concentration of 30 μg/mL lysozyme and 1% PMSF, sonicated on ice for 30 min, and centrifuged at 12,000 rpm. After 40 min, the supernatant was collected. The supernatant was added to the amylose resin affinity chromatography column, and the column was washed with 10 column volumes of wash buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 2 mM maltose), and finally 10 A column volume of elution buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM maltose) buffer was used for elution. The protein concentration was determined by Nanodrop 2000, and it was calculated that 35.5 mg of wild-type αSN protein could be obtained per liter of bacterial solution.

For the expression of pAzF mutant protein, the helper plasmid pEVOL-pAzF was co-transformed with the expression vector plasmid pET22b-mutαSN (mutαSN are 6 mutant protein encoding genes, pET22b-mutαSN represents the pET22b expression vector carrying the αSN mutant protein encoding gene) The same E. coli BL21 competent cells were used to construct the corresponding recombinant strain BL21-mutαSN. Pick a single colony of the above-mentioned engineered bacteria to 5mL LB medium for overnight culture at 37°C, transfer to fresh LB medium at 1% inoculum size, cultivate at 37°C to OD600 of 0.3, and add a final concentration of 1mM pAzF. Continue to be cultured in a shaker at 37°C, when the OD600 reaches 0.5, add a final concentration of 0.2% (w/v) L-arabinose, and place it in a shaker at 30°C to culture until the OD600 is 1, add a final concentration of 0.5mM IPTG Induction, the induction temperature was 30 °C, and the induction time was 16-18 h. Finally, the cells were collected by centrifugation at 6000 rpm for 10 min. Consistent with the above-mentioned protein purification method, 6 kinds of pAzF mutant proteins were obtained by Ni-column affinity chromatography. The protein concentration was detected by Nanodrop 2000, and the yields of the six mutant proteins αSN4F, αSN39Y, αSN94F, αSN125Y, αSN133Y, and αSN136Y were calculated to be approximately 7.5 mg/L, 13.7 mg/L, 8.3 mg/L, 8.8 mg/L, and 9.1 mg, respectively. /L, 7.8mg/L. The above proteins were lyophilized after desalting by dialysis, and placed at -20°C for later use.

MALDI TOF mass spectrometry was used to further verify that the obtained protein was the target protein, and the mass spectrogram is shown in Figure 4.

Example 3: ThT fluorescent staining to detect the aggregation properties of mutant proteins

The ex-situ culture method was used for detection. The above-mentioned six mutant proteins and αSN were respectively prepared into equal concentration protein solutions with a concentration of 100 μM, placed at 37° C. and incubated with shaking at 180 rpm. Sampling at a fixed time, the sampling volume is 10 μL, 90 μL of 250 μM ThT solution is added, and the fluorescence intensity is detected under the conditions of excitation wavelength 440 nm and emission wavelength 480 nm after thorough mixing. The detection results are shown in Figure 5. It can be seen from the figure that with the increase of culture time, the fluorescence intensity of wild-type αSN gradually increased, and basically entered a stable phase after 12 hours; among the six mutant proteins, the fluorescence intensity of αSN4F and αSN39Y also increased with time. The fluorescence intensity of the other four mutant proteins was always low. The above results prove that when pAzF is introduced into positions 4 and 39, it has little effect on the aggregation of the protein itself. When the amino acids at the other 4 positions are replaced, the protein basically loses the aggregation. Therefore, two mutant proteins, αSN4F and αSN39Y, were selected for follow-up. experiment of.

The above protein and ThT buffer were TBS buffer, pH 5.0.

Example 4: Construction of EPB-αSN4F/αSN39Y system

The present invention adopts copper-catalyzed azide-terminal alkyne cycloaddition reaction (CuAAC) to connect EPB with αSN4F or αSN39Y to construct an EPB-αSN4F or EPB-αSN39Y system, as shown in Figure 6-a. 1mL reaction system, reaction conditions and the amount of reagents used are as follows:

Then make up 1 mL with PBS. Mix and shake gently, and react at 4°C for 8 hours (the above buffers and reagents are all prepared with PBS). After the reaction is completed, the reaction system is desalted by using a dialysis bag or a desalting column to remove excess ions, etc., and the dialysis or desalination process should be kept as low as possible.

In order to verify whether the above system was successfully constructed, the UV-Vis spectrophotometer was used to scan in the wavelength range of 200-600nm to compare and detect the changes in the absorbance of the target protein before and after EPB labeling. The results are shown in Figures 6-b and 6-c. Show. It is obvious from the figure that the absorbance of EPB-αSN4F or EPB-αSN39Y complexes after EPB labeling in the wavelength range of 250–350 nm is significantly larger than that of unlabeled αSN4F or αSN39Y. This phenomenon is due to the π-π on EPB. The bond resulted in an increase in absorbance, demonstrating the successful attachment of EPB to αSN4F or αSN39Y.

Example 5: Application of EPB-αSN4F or EPB-αSN39Y system

In order to identify whether the system can really be used in the screening of aggregation inhibitors, EGCG, a small molecule inhibitor known to have αSN aggregation inhibitory effect, was selected for verification.

EGCG powder was formulated to a concentration of 100 [mu]M stock solution in PBS buffer. The same final concentration (20 μM) of EGCG and EPB-αSN4F or EPB-αSN39Y solution was placed in a 96-well cell culture plate to prepare a 200 μL system. After fully mixing, use a fluorescence spectrophotometer to scan and detect, and the detection conditions are: excitation wavelength 350nm, emission wavelength 380nm-600nm. The same final concentration of EPB-αSN4F or EPB-αSN39Y without EGCG was used as a control group.

The experimental results are shown in Figure 7. As a blank control, the fluorescence value of EGCG solution in this scanning range, especially in the range of 450-550nm, is extremely low, while the fluorescence value of EPB-αSN4F or EPB-αSN39Y solution without EGCG is significantly higher than that of EPB-αSN4F solution without EGCG. Add samples of EGCG. It is proved that because EGCG inhibits the aggregation of the target protein, the aggregation-induced luminescence system is in a free state, so the fluorescence value is low. When EGCG is not added, the target protein forms aggregates and finally induces the system to emit strong fluorescence. In addition, the analysis results show that within this scanning range, the fluorescence value is the highest when the emission wavelength is 490 nm, which can be used as a detection condition for rapid screening of small molecule drugs in the later stage.

Example 6 Study of Aggregation Properties

(1) Construction of a luminescence system ① αSN and EPB solution were fully mixed at equal concentrations, and the final concentration was 20 μM; ② αSN4F or αSN39Y and EPB were fully mixed at equal concentrations, and the final concentration was 20 μM; ③ 20 μM EPB-αSN4F or EPB-αSN39Y;

(2) Scanning and detecting the above systems using a fluorescence spectrophotometer respectively, and the detection conditions are: excitation wavelength of 350 nm and emission wavelength of 380 nm-600 nm.

The results are shown in FIG. 8 . It can be seen from FIG. 8 that EPB cannot generate fluorescence with the aggregation of αSN, that is, EPB cannot be used alone to study the aggregation properties of αSN. The αSN mutated by pAzF also cannot make EPB aggregate to generate fluorescence during the aggregation process, while the EPB-αSN4F or EPB-αSN39Y constructed in the present invention can induce the system to emit strong fluorescence when forming aggregates.

To sum up, the aggregation-induced luminescence system constructed in the present invention can be applied to the screening of αSN aggregation inhibitors. When the inhibitor exists in the solution, the αSN aggregation can be inhibited, resulting in the sensor not emitting light; When an inhibitor is present, αSN rapidly aggregates, so that the sensor can emit strong fluorescence, which can be detected by a fluorescence spectrophotometer, and the inhibitory effect of the small molecule inhibitor is determined according to the fluorescence intensity.

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Claims (6)

1. an αSN aggregation-induced luminescent fusion, is characterized in that, the αSN aggregation-induced luminescent fusion is specifically EPB-αSN39Y, which is to replace the tyrosine at position 39 in the αSN amino acid sequence with p-azidophenylpropane. amino acid pAZF to obtain αSN39Y mutant protein, and then bio-orthogonal ligation reaction of AIE molecule EPB and mutant protein αSN39Y to obtain EPB-αSN39Y; The αSN amino acid sequence is shown in SEQ ID No.2 of the sequence table; The chemical formula of the EPB is: C ₃₈ H ₄₄ N ₂ O ₂ Br ₂ . 2. the construction method of the described αSN aggregation-induced luminescence fusion of claim 1, is characterized in that, is specifically as follows: (1) Obtain a protein expression gene that replaces the tyrosine at position 39 in αSN with pAZF by gene synthesis or PCR, and obtain a αSN39Y mutant protein by expressing in E. coli; (2) Using copper-catalyzed azide-terminal alkyne cycloaddition reaction, EPB and mutant protein αSN39Y were bio-orthogonally linked to obtain EPB-αSN39Y. 3. the construction method of αSN aggregation-induced luminescence fusion as claimed in claim 2, is characterized in that, adopts copper-catalyzed azide terminal alkyne cycloaddition reaction to connect EPB and αSN39Y, and the method for constructing EPB-αSN39Y screening system is as follows: 1mL reaction system, reaction conditions and the amount of reagents used are as follows:

Then, PBS was used to make up 1 mL; the reaction was performed at 4° C. for 8 h. After the reaction was completed, the reaction system was desalted with a dialysis bag or a desalting column. 4. The application of the αSN aggregation-induced luminescent fusion of claim 1 in the screening of αSN aggregation inhibitors. 5. application as claimed in claim 4 is characterized in that, be specifically applied to screening αSN aggregation inhibitor, the method is as follows: in EPB-αSN39Y solution, add potential inhibitor, excitation wavelength 350nm, emission wavelength 380nm-600nm range detection If the fluorescence intensity is weaker than that of the EPB-αSN39Y system without a potential inhibitor, the potential inhibitor is determined to be an αSN aggregation inhibitor. 6 . The application according to claim 5 , wherein the detection is performed under the condition that the concentration of EPB-αSN39Y is 20 μM, the excitation wavelength is 350 nm, and the emission wavelength is 490 nm. 7 .

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