CN108642028B

CN108642028B - Alkalized lysozyme for inhibiting beta amyloid protein aggregation and preparation method and application thereof

Info

Publication number: CN108642028B
Application number: CN201810304964.3A
Authority: CN
Inventors: 孙彦; 李茜; 史清洪; 董晓燕
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2018-04-08
Filing date: 2018-04-08
Publication date: 2021-11-19
Anticipated expiration: 2038-04-08
Also published as: CN108642028A

Abstract

The invention relates to alkalized lysozyme for inhibiting beta amyloid protein aggregation and a preparation method and application thereof, wherein the average modification number on carboxyl on the surface of lysozyme molecules is between 0.5 and 7 ethylenediamine molecules. Adding ethylenediamine liquid into 0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution, adjusting the pH value to 4.5-7.5 by using concentrated hydrochloric acid, adding lysozyme solid for dissolving, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and stirring for reaction; and removing free ethylenediamine and EDC to obtain the basified lysozyme with a stable molecular structure. The alkalized lysozyme is synthesized by chemically modifying the surface of the lysozyme, so that the positive charge on the surface of the lysozyme is effectively increased, and the reaction is mild, simple and convenient. The alkalized lysozyme can effectively inhibit the aggregation of the A beta 42, change the morphology of the A beta 42 aggregate, and prevent and slow down the transformation to fibrous morphology. Has wide application prospect as a medicine for treating Alzheimer's disease.

Description

Alkalized lysozyme for inhibiting beta amyloid protein aggregation and preparation method and application thereof

Technical Field

The invention relates to alkalized lysozyme for inhibiting beta amyloid protein aggregation and a preparation method and application thereof, belonging to the technical field of biological medicines.

Background

Alzheimer's Disease (AD) is the most prominent form of senile dementia, with over 4700 million AD patients worldwide by 2016, and this figure is expected to reach 1 million 3100 million by 2050 (World Alzheimer report 2016). The pathological features of AD are kinking of nerve fibers within nerve cells and extracellular senile plaque deposition (Nature,2014,515: 274-. The main component of senile plaques is amyloid beta (a β). A beta is a polypeptide comprising 39-43 amino acid residues, which is cleaved from Amyloid Precursor Protein (APP) by beta and gamma secretases in sequence (Langmuir,2012,28: 6595-. The two main types of A β are A β 40 and A β 42, respectively, in which A β 40 is more abundant and A β 42 is more toxic (Science,2006,314: 777-. Studies have shown that aggregation of A.beta.according to the nucleus-dependent growth process, A.beta.first forms a nucleus with a regular structure, and then oligomers in solution bind to the nucleus and grow further into fibrils, fibers (Journal of molecular biology,2012,422: 723-730). In conformation, the natural monomer A beta presents a random coil structure, and when aggregation occurs, the A beta conformation is gradually converted into a beta-sheet structure, and the A beta with the beta-sheet conformation is more favorable for the formation of amyloid aggregates. Based on the amyloid hypothesis, amyloid aggregation of A β and its resulting neurotoxicity are the main causes of pathological features in AD, and thus inhibition of amyloid aggregation of A β is a viable means of treating AD, capable of preventing or delaying the onset of AD (Current opinion in structural biology,2003,13: 526-.

At present, the reported types of a β inhibitors include small molecules, polypeptides, nanoparticles and proteins. Small molecule inhibitors are simple in structure and easy to obtain, but most of them have the defects of poor solubility, short half period in vivo, poor stability, low bioavailability and the like (Journal of Biological Chemistry,2007,282: 10311-. Although polypeptide inhibitors have the advantages of good biocompatibility, small molecular weight and the like, the polypeptide inhibitors have general inhibition effects and have serious defects of self-aggregation tendency, poor in-vivo stability, easy protease hydrolysis and the like (ACS Chemical Neuroscience,2010,1: 661-. In recent years, nanoparticles have the characteristics of simple synthesis, easy functionalization, large specific surface area and the like, and show great advantages in the development of A beta aggregation inhibitors, however, some nanoparticle inhibitors have the defects of poor biocompatibility, general inhibition effect and the like (Chemical Science,2012,3: 868-.

Compared with the inhibitors, protein inhibitors are increasingly paid more attention because of the advantages of good biocompatibility, convenience in chemical modification and the like. Human lysozyme (hLys) is an important natural non-specific immune protein, widely present in body fluids and tissues (Scientific reports,2016,6: 22947). In the brains of AD patients, hLys was found to be present in amyloid plaques together with A β (Neurobiology of disease 2015,83: 122-133). It has also been reported that hLys is effective in inhibiting aggregation and toxicity of A β 1-40, A β 1-42 and A β 17-42 (Chemical Communications,2013,49: 6507-. However, the inhibitory effect of hLys is still limited. Therefore, how to improve the ability of hLys to inhibit A β aggregation becomes an important technical problem for developing candidate drugs for AD therapy.

Studies have shown that under physiological conditions, A.beta.is negatively charged and its interaction with lysozyme is dominated by electrostatic and hydrophobic interactions (Chemical Communications,2013,49: 6507-. Therefore, the invention provides a method for inhibiting aggregation of Abeta more effectively by chemically modifying the surface of hLys, eliminating negative charges, introducing positive charges, and obtaining stronger electrostatic interaction between alkalinized lysozyme and Abeta under physiological conditions.

Disclosure of Invention

The invention aims to provide an alkalizing lysozyme inhibitor with a function of inhibiting beta-amyloid protein aggregation, a preparation method and application thereof in inhibiting beta-amyloid protein aggregation. The alkalized lysozyme inhibitor has stable structural property and good biocompatibility, and has good inhibition effect on A beta aggregation and toxicity.

The invention is realized by the following technical scheme,

an alkalized lysozyme is characterized in that the average modification number on carboxyl on the surface of lysozyme molecules is between 0.5 and 7 ethylenediamine molecules. The alkalized lysozyme has the following structure:

black ball

Represents a lysozyme molecule.

Adding ethylenediamine liquid into 0.1 mol/L2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, then adjusting the pH value to 4.5-7.5 by using concentrated hydrochloric acid, then adding lysozyme solid to dissolve the lysozyme solid, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and stirring for reaction; free ethylenediamine and EDC were removed to give basified lysozyme (B-hLys) with a stable molecular structure.

The concentration of lysozyme solid added is preferably 1-20 mg/mL.

Preferably, the molar ratio of the ethylenediamine to the lysozyme added into the 2- (N-morpholine) ethanesulfonic acid buffer solution (namely the adding amount of the ethylenediamine) is 5-400: 1.

The concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride added is preferably 1-20 mg/mL.

The preferable stirring reaction condition is that the stirring reaction is carried out for 0.5 to 24 hours at the temperature of between 25 and 50 ℃.

The reaction formula of the invention is as follows:

the invention synthesizes alkalized lysozyme, the mass spectrum is utilized to measure the average molecular weight, 0.5-7 ethylenediamine molecules are modified on the surface of the lysozyme through calculation, and zeta potentials of the lysozyme before and after reaction are tested through experiments, the results show that the reaction can effectively increase positive charges on the surface of the lysozyme under physiological conditions, and particle size and endogenous fluorescence experiments show that the modified alkalized lysozyme keeps the structure and stability of natural lysozyme.

The alkalized lysozyme synthesized by the invention enhances the interaction with the A beta, and compared with the A beta cultured alone, the alkalized lysozyme with 0.5-fold molar weight reduces the ThT fluorescence value from 100% to 19.0%, and reduces the cytotoxicity generated by the A beta aggregate from 37.5% to 8.6%. Atomic Force Microscopy (AFM) observations indicate that the Α β morphology co-cultured with alkalinized lysozyme was transformed from long fibrous aggregates to small amorphous aggregates. Therefore, the basified lysozyme can effectively inhibit the aggregation of A beta and reduce the toxicity of the A beta aggregate on cells.

The basified lysozyme can inhibit A beta aggregation and reduce the cytotoxicity of the A beta aggregation by using the basified lysozyme in the application of inhibiting and preventing the beta-amyloid aggregation, so that the basified lysozyme can be used for treating diseases related to the beta-amyloid aggregation.

The basified lysozyme is applied to protein medicaments, and the protein medicaments can be used for preventing, treating and delaying diseases such as Alzheimer disease and the like.

The invention provides a novel alkalization lysozyme inhibitor with a function of inhibiting beta-amyloid aggregation, a preparation method and application thereof in inhibiting beta-amyloid aggregation, and the inhibitor has the following advantages:

firstly, the alkalized lysozyme is synthesized by chemically modifying the surface of the lysozyme, so that the positive charge on the surface of the lysozyme can be effectively increased, and the reaction is mild and simple.

Second, the alkalized lysozyme is able to effectively inhibit the aggregation of a β 42, which alters the morphology of a β 42 aggregates, preventing and slowing down their transformation to fibrous morphology.

Thirdly, the basifying lysozyme effectively reduces the toxicity of the A beta 42 aggregate on cells, and is an ideal inhibitor of the A beta 42 aggregate. Has wide application prospect as a medicine for treating Alzheimer's disease.

Drawings

FIG. 1: endogenous fluorescence profiles of lysozyme and low modified density alkalinized lysozyme (B-hLys1) at the same concentration in example 1.

FIG. 2: endogenous fluorescence change pattern of the same concentration of lysozyme and medium modified density of alkalinized lysozyme (B-hLys2) in example 2.

FIG. 3: endogenous fluorescence change patterns of lysozyme and alkalized lysozyme with high modification density (B-hLys3) in the same concentration in example 3.

FIG. 4: ThT fluorescence profiles of cultures incubated with A β 42 for 24h with B-hLys at different modification densities in example 4.

FIG. 5: ThT fluorescence profiles of cultures incubated with A β 42 for 24h at different concentrations of B-hLys in example 5.

FIG. 6: AFM images of cultures after 24h co-cultivation of alkalinized lysozyme with A β 42 in example 6.

FIG. 7: example 7 cell viability of cultures on SH-SY5Y after 24h co-culture of alkalinized lysozyme with A β 42.

Detailed Description

The present invention will be further described with reference to the following specific examples.

The invention carries out chemical modification on the surface of the lysozyme, thereby improving the positive charge carried on the surface of the lysozyme and preparing the alkalized lysozyme. A plurality of experimental means prove that the alkalinizing lysozyme can effectively inhibit the aggregation of A beta 42 and can reduce the cytotoxicity generated in the aggregation process of the A beta 42. The ability of alkalinizing lysozyme to inhibit a β 42 aggregation was determined by methods such as ThT fluorescence and atomic force microscopy, and its ability to reduce cytotoxicity generated during a β 42 aggregation was determined by MTT cytotoxicity assay.

Example 1: synthesis and characterization of alkalized lysozyme with molar ratio of ethylenediamine to lysozyme of 5

10ml of MES buffer (0.1mol/L), 0.2. mu.L of ethylenediamine liquid (molar ratio of ethylenediamine to lysozyme: 5) was added to the buffer, pH was adjusted to 7.5 using concentrated hydrochloric acid, 10mg of lysozyme solid was added to dissolve it, and finally 1mg of EDC solid was added. The mixture was stirred at 170rpm and 25 ℃ for 0.5 h. Dialyzing to remove free ethylenediamine and EDC in the system, wherein the dialysate is water, and the obtained product: the basified lysozyme with low modification density (B-hLys1) is stored after being frozen and dried.

Quantitative hLys and B-hLys1 were weighed and dissolved in a Phosphate Buffer Solution (PBS) at pH 7.4 (phosphate buffer saline) to a concentration of 1mg/ml, wherein the phosphate concentration was 100mmol/L and the NaCl concentration was 10mmol/L, respectively. The change in molecular size of lysozyme before and after modification was analyzed by a particle size analyzer (Malvern Instruments) at 25 ℃; the Zeta potential of the same concentrations of hLys and B-hLys1 was determined by potentiometry (Malvern Instruments), respectively; the molecular weight of B-hLys1 was analyzed by mass spectrometer to determine the average modification rate of B-hLys 1. And finally, measuring the endogenous fluorescence intensity change of the lysozyme under the excitation wavelength of 286nm by using a fluorescence spectrophotometer, and comparing the change of the molecular structure of the lysozyme before and after modification.

TABLE 1 characterization of the physicochemical properties of hLys and B-hLys1 at pH 7.4

As is clear from Table 1, the particle size of hLys is 4.6 + -0.3 nm, while the particle size of B-hLys1 is 4.7 + -0.2 nm, indicating that the particle size of lysozyme after modification does not change much. The zeta potential of B-hLys1 (15.2. + -. 0.7mV) was higher than that of hLys (12.4. + -. 0.5mV) at pH 7.4, indicating that the modification of ethylenediamine reduced the carboxyl groups and increased the amino groups on the surface of hLys, thus increasing its zeta potential. The molecular weight of B-hLys1 is 14718.713Da, the molecular weight of natural lysozyme is 14697.148 Da, and the average modification rate of B-hLys1 is 0.5 ethylenediamine/lysozyme due to the fact that one ethylenediamine has the molecular weight of 60.12 and one water molecule is removed.

As shown in FIG. 1, the endogenous fluorescence intensity and the peak position of hLys and B-hLys1 are basically consistent under 286nm excitation, which shows that the spatial structure of the modified lysozyme is still kept compact and basically consistent with the natural state.

The results show that the modification method successfully modifies ethylenediamine on the surface of lysozyme and increases the positive charge on the surface of lysozyme on the premise of ensuring that the spatial structure and the stability of protein are not changed.

Example 2: synthesis and characterization of alkalized lysozyme with molar ratio of ethylenediamine to lysozyme of 220

10ml of MES buffer (0.1mol/L) was taken, 90. mu.L of ethylenediamine liquid (molar ratio of ethylenediamine to lysozyme: 220) was added to the buffer, pH was adjusted to 6.0 using concentrated hydrochloric acid, and 100mg of lysozyme solid was added to dissolve it, and finally 50mg of EDC solid was added. The mixture was stirred at 170rpm and 37 ℃ for 12 h. Dialyzing to remove free ethylenediamine and EDC in the system, wherein the dialysate is water, and the obtained product: the medium modified density alkalized lysozyme (B-hLys2) is stored after being frozen and dried.

Quantitative hLys and B-hLys2 were weighed and dissolved in a Phosphate Buffer Solution (PBS) at pH 7.4 (phosphate buffer saline) to a concentration of 1mg/ml, wherein the phosphate concentration was 100mmol/L and the NaCl concentration was 10mmol/L, respectively. The change in molecular size of lysozyme before and after modification was analyzed by a particle size analyzer (Malvern Instruments) at 25 ℃; the Zeta potential of the same concentrations of hLys and B-hLys2 was determined by potentiometry (Malvern Instruments), respectively; the molecular weight of B-hLys2 was analyzed by mass spectrometer to determine the average modification rate of B-hLys 2. And finally, measuring the endogenous fluorescence intensity change of the lysozyme under the excitation wavelength of 286nm by using a fluorescence spectrophotometer, and comparing the change of the molecular structure of the lysozyme before and after modification.

TABLE 2 characterization of the physicochemical properties of hLys and B-hLys2 at pH 7.4

As is clear from Table 2, the particle size of hLys is 4.6 + -0.3 nm, while the particle size of B-hLys2 is 4.8 + -0.3 nm, indicating that the particle size of lysozyme after modification does not change much. The zeta potential of B-hLys2 (23.3. + -. 0.2mV) was higher than that of hLys (12.4. + -. 0.5mV) at pH 7.4, indicating that the modification of ethylenediamine reduced the carboxyl groups and increased the amino groups on the surface of hLys, thus increasing its zeta potential. The molecular weight of B-hLys2 is 14874.052Da, the molecular weight of natural lysozyme is 14697.148 Da, and the average modification rate of B-hLys2 is 4 ethylenediamine/lysozyme because one ethylenediamine has the molecular weight of 60.12 and one water molecule is removed.

As shown in FIG. 2, the endogenous fluorescence intensity and the peak position of hLys and B-hLys2 are basically consistent under 286nm excitation, which shows that the spatial structure of the modified lysozyme is still kept compact and basically consistent with the natural state.

Example 3: synthesis and characterization of alkalized lysozyme with molar ratio of ethylenediamine to lysozyme of 400

10ml of MES buffer (0.1mol/L) was taken, 327. mu.L of ethylenediamine liquid (molar ratio of ethylenediamine to lysozyme: 400) was added to the buffer, pH was adjusted to 4.7 using concentrated hydrochloric acid, 200mg of lysozyme solid was added to dissolve it, and finally 100mg of EDC solid was added. The mixture was stirred at 170rpm and 50 ℃ for 24 h. Dialyzing to remove free ethylenediamine and EDC in the system, wherein the dialysate is water, and the obtained product: the basified lysozyme with high modification density (B-hLys3) is stored after being frozen and dried.

Quantitative hLys and B-hLys3 were weighed and dissolved in a Phosphate Buffer Solution (PBS) at pH 7.4 (phosphate buffer saline) to a concentration of 1mg/ml, wherein the phosphate concentration was 100mmol/L and the NaCl concentration was 10mmol/L, respectively. The change in molecular size of lysozyme before and after modification was analyzed by a particle size analyzer (Malvern Instruments) at 25 ℃; the Zeta potential of the same concentrations of hLys and B-hLys3 was determined by potentiometry (Malvern Instruments), respectively; the molecular weight of B-hLys3 was analyzed by mass spectrometer to determine the average modification rate of B-hLys 3. And finally, measuring the endogenous fluorescence intensity change of the lysozyme under the excitation wavelength of 286nm by using a fluorescence spectrophotometer, and comparing the change of the molecular structure of the lysozyme before and after modification.

TABLE 3 characterization of the physicochemical properties of hLys and B-hLys3 at pH 7.4

As is clear from Table 3, the particle size of hLys is 4.6 + -0.3 nm, while the particle size of B-hLys3 is 4.9 + -0.2 nm, indicating that the particle size of lysozyme after modification does not change much. The zeta potential of B-hLys3 (32.1 + -0.6 mV) was higher than that of hLys (12.4 + -0.5 mV) at pH 7.4, indicating that the modification of ethylenediamine reduced the carboxyl groups and increased the amino groups on the surface of hLys, thereby increasing its zeta potential. The molecular weight of B-hLys3 is 14994.936Da, the molecular weight of natural lysozyme is 14697.148 Da, and the average modification rate of B-hLys3 is 7 ethylenediamine/lysozyme because one ethylenediamine has the molecular weight of 60.12 and one water molecule is removed.

As shown in FIG. 3, the endogenous fluorescence intensity and the peak position of hLys and B-hLys3 are basically consistent under 286nm excitation, which indicates that the spatial structure of the modified lysozyme is still kept compact and basically consistent with the natural state.

Example 4: ThT fluorescence intensity of cultures after incubation of different modified densities of alkalinized lysozyme with A β 42 for 24 h.

The effects of different modified densities of alkalinized lysozyme on the fluorescence intensity of A.beta.42 ThT were examined using B-hLys1, B-hLys2, and B-hLys3 synthesized in examples 1, 2, and 3.

Dissolving Abeta 42 with purity of 95% in Hexafluoroisopropanol (HFIP) (1.0-1.5mg/ml), ultrasonic treating for 10min to break aggregate, standing to dissolve completely, centrifuging at 4 deg.C and 16000g for 30min, and freezing 75% of supernatant in-70 deg.C refrigerator. Finally, putting the mixture into a freeze dryer, freeze-drying the mixture for 24 hours, and storing the mixture in a refrigerator at the temperature of-20 ℃.

Dissolving the Abeta 42 in a 20mmol/L NaOH solution, and performing ultrasonic treatment for 15min to fully dissolve the Abeta 42 to obtain an Abeta 42 mother solution with the concentration of 275 mu mol/L. The solution was diluted with 100mmol/L PBS buffer (pH 7.4, containing 10mmol/L NaCl) to obtain a final concentration of 25. mu. mol/L of A.beta.42 solution as a control experiment.

B-hLys1, B-hLys2, B-hLys3 and hLys were weighed and dissolved in 100mmol/L PBS buffer (pH 7.4, containing 10mmol/LNaCl) to obtain protein solutions with different modification densities at a concentration of 2.75. mu. mol/L. A beta 42 mother liquor with the concentration of 275 mu mol/L is taken and diluted by four protein solutions with the concentration of 2.75 mu mol/L respectively to obtain the A beta 42 with the final concentration of 25 mu mol/L and the protein solution with the concentration of 2.5 mu mol/L. The four solutions were incubated at 37 ℃ for 24h at 150rpm, along with the control solution.

3.99mg of ThT was dissolved in 500mL of 100mmol/L PBS buffer (pH 7.4, containing 10mmol/L NaCl) to prepare a ThT solution having a final concentration of 25. mu. mol/L. 200 mu L of Abeta 42 sample cultured for 24h is mixed with 2mL of 25 mu mol/L ThT solution, the fluorescence intensity is detected under the excitation wavelength of 440nm and the emission wavelength of 480nm, the excitation and emission slit width is 5nm, the scanning speed is 100nm/min, and the scanning results are average values of 3 times. The fluorescence intensity at 480nm was plotted against time. The results are shown in FIG. 4.

As can be seen from FIG. 4, the inhibition effect of B-hLys1, B-hLys2 and B-hLys3 on A beta 42 aggregation is gradually enhanced compared with hLys, which shows that the average modification rate of the ethylene diamine modified by the alkalized lysozyme is in the range of 0.5-7, the inhibition effect on A beta 42 aggregation is obviously improved compared with natural lysozyme, and the effect is gradually enhanced along with the increase of the average modification rate.

Example 5: ThT fluorescence intensity of the cultures after 24h co-culture of different concentrations of alkalinized lysozyme with A β 42.

The effect of different concentrations of alkalinizing lysozyme on ThT fluorescence intensity of A β 42 aggregates was examined with B-hLys3 synthesized in example 3.

A ThT solution, Abeta 42 stock, was prepared as in example 4. The A.beta.42 stock solution was diluted with 100mmol/L PBS buffer (pH 7.4, containing 10mmol/L NaCl) to obtain a final concentration of 25. mu. mol/L of A.beta.42 solution as a control experiment.

B-hLys3 and hLys were dissolved in 100mmol/L PBS buffer (pH 7.4, containing 10mmol/L NaCl) to obtain protein solutions with concentrations of 2.75, 6.875 and 13.75. mu. mol/L, respectively. A beta 42 mother liquor with the concentration of 275 mu mol/L is taken and diluted by protein solutions with different concentrations respectively to obtain protein solutions with the final concentrations of 2.5, 6.25 and 12.5 mu mol/L and 25 mu mol/L A beta 42 solutions respectively. These solutions of different concentrations were incubated for 24h at 37 ℃ and 150rpm, together with the control solution. 200 mu L of Abeta 42 sample cultured for 24h is mixed with 2mL of 25 mu mol/L ThT solution, the fluorescence intensity is detected under the excitation wavelength of 440nm and the emission wavelength of 480nm, the excitation and emission slit width is 5nm, the scanning speed is 100nm/min, and the scanning results are average values of 3 times. The fluorescence intensity at 480nm was plotted against time. As shown in fig. 5.

As can be seen from FIG. 5, the inhibition of A beta 42 aggregation by lysozyme and basified lysozyme is concentration-dependent, i.e., the inhibition effect is gradually increased with the increase of the concentration. The effect of the alkalized lysozyme is obviously better than that of the natural lysozyme under the condition of the same concentration.

Example 6: the basified lysozyme was co-cultured with Abeta 42 for 24 h.

The effect of alkalinizing lysozyme on the morphology of A β 42 aggregates was examined using B-hLys3 synthesized in example 3.

A β 42 sample was prepared in the same manner as in example 4. 2 types of A beta 42 samples are prepared, and respectively comprise: the A beta 42 solution and the A beta 42 solution containing 12.5 mu mol/L B-hLys3, wherein the final concentration of the A beta 42 in the solution is 25 mu mol/L. The above solution was incubated at 37 ℃ and 150 rpm.

After 24h incubation, 10. mu.L of the suspension was dropped onto a clean mica plate and allowed to settle for 5 min. Washing the substrate with deionized water several times, washing to remove salt ions in the solution, and naturally drying or drying with nitrogen. Observed with a multimode atomic force microscope (CSPM5500, primitive) and imaged in tapping mode.

As can be seen from FIG. 6A, A.beta.42 alone exhibited a long and dense reticular fibrous shape after 24 hours of culture. After the co-culture of B-hLys and A beta 42 in FIG. 6B, the morphology is changed, and small and unshaped aggregates are formed instead of fibers, which shows that the B-hLys can effectively inhibit the aggregation of A beta 42 and change the aggregation morphology of A beta 42.

Example 7: the cell survival rate of the culture on SH-SY5Y cells after the basified lysozyme and the Abeta 42 are co-cultured for 24 hours.

The effect of alkalinizing lysozyme on A β 42 aggregate cytotoxicity was examined using B-hLys3 synthesized in example 3.

The cells used in the cytotoxicity test were human neuroblastoma cell line (SH-SY 5Y). The culture medium is DMEM/F-12 medium, containing 20% FBS, 2mmol/L glutamine, and 100U/mL double antibody. The culture environment is 5% CO₂Culturing at 37 deg.C.

In the MTT assay, cells were added to a 96-well plate at a density of about 5000 cells per 90 μ L of culture medium per well. After 24h incubation, the aged Abeta 42 or a sample co-incubated with alkalinized lysozyme (10. mu.L) was added to achieve a final concentration of 25. mu. mol/L lysozyme and 25. mu. mol/L Abeta 42 in the wells. After further incubation for 24h, 10. mu.L of MTT solution (5.5 mg/mL) was added to each well and incubation was continued for 4 h. During detection, a 96-well plate is centrifuged at 1500rpm for 10min, the solution in the 96-well plate is removed after centrifugation, and 100 μ L of DMSO is added into each well and shaken on a shaker for 10 min. After formazan was completely dissolved, absorbance was measured at 570nm using an enzyme reader, and the cell survival rate was calculated. Cell-free samples were used as a blank (0%); cell viability was calculated for the drug-added group as a control group (100%) for samples containing only cells without a β and inhibitor. Each sample was set up with 6 duplicate wells in the experiment. The results are shown in FIG. 7.

As can be seen from FIG. 7, hLys and B-hLys do not have cytotoxicity, the A beta 42 aggregate can kill about 40% of cells, the cell survival rate is obviously improved after the hLys or the B-hLys is added, and the B-hLys effect is more obvious, which shows that the B-hLys can effectively inhibit the cytotoxicity generated by the A beta 42 aggregate.

The invention provides a technology for preparing the alkalized lysozyme with good stability and biocompatibility by coupling ethylenediamine on the surface of human lysozyme through a chemical modification method so as to improve the positive charge on the surface of the human lysozyme. The application of the basified lysozyme in the preparation of medicines for inhibiting the aggregation of beta-amyloid protein and the like is provided, and the basified lysozyme is applied to conformational change, aggregation and cytotoxicity inhibition experiments of A beta 42. Having described preferred embodiments in the field, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced with modification, or with appropriate modification and combination, of the methods described herein without departing from the spirit, scope, and spirit of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.

Claims

1. An alkalized lysozyme for inhibiting beta amyloid protein aggregation, which is characterized in that the average modification number on carboxyl on the surface of lysozyme molecules is between 0.5 and 7 ethylenediamine molecules; the alkalized lysozyme has the following structure:

lysozyme black ball

Represents human lysozyme.

2. A process for preparing alkalinizing lysozyme for inhibiting amyloid beta aggregation according to claim 1, characterized in that: adding ethylenediamine liquid into 0.1 mol/L2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, adjusting the pH value to 4.5-7.5 by using concentrated hydrochloric acid, then adding lysozyme solid to dissolve the lysozyme solid, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and stirring for reaction; and removing free ethylenediamine and EDC to obtain the basified lysozyme with a stable molecular structure.

3. The method of claim 2, wherein the concentration of lysozyme solid is 1 to 20 mg/mL.

4. The method according to claim 2, wherein the molar ratio of ethylenediamine to lysozyme added to the 2- (N-morpholino) ethanesulfonic acid buffer is in the range of 5 to 400: 1.

5. The method according to claim 2, wherein the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 to 20 mg/mL.

6. The method as set forth in claim 2, wherein the stirring reaction is carried out at 25-50 ℃ for 0.5-24 hours.

7. Use of the basified lysozyme of claim 1 for the preparation of a pharmaceutical material having the effect of inhibiting the aggregation of β -amyloid.

8. Use of the basified lysozyme of claim 1 for the preparation of a pharmaceutical material having reduced cytotoxicity of β -amyloid aggregates.

9. Use of the basified lysozyme of claim 1 for the preparation of a pharmaceutical raw material for the treatment of alzheimer's disease.