Polypeptide sequence and preparation method of exosomes for treating depression

Abstract

The present invention provides a polypeptide sequence, a polyclonal antibody mixture obtained by immunizing an animal with the polypeptide sequence, a latex microsphere prepared by the polyclonal antibody mixture, and the use of the latex microsphere in extracting and separating exosomes for the treatment of depression. Preferably, the exosomes are exosomes for separating depression. The present invention also provides a method for preparing exosomes for the treatment of depression. The present invention obtains polyclonal antibodies with a high affinity for cell exosomes, and uses them as core raw materials to make latex microspheres for exosome separation and preparation. By optimizing the affinity of the coated antibodies of the latex microspheres, the efficiency of exosome separation and preparation is improved.

Description

Technical Field

[0001] The present invention relates to a polypeptide sequence and a method for preparing exosomes for treating depression. Background Art

[0002] Exosomes are membrane vesicles that are released into the extracellular matrix after the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane. They are generally 40-100nm in diameter. The lipid bilayer membrane encapsulates substances such as DNA, RNA and proteins. Exosomes have a variety of functions. Almost all types of cells can produce and release exosomes. Depending on the cell type from which they are derived, exosomes can affect the body's cell migration, cell differentiation, tumor invasion, cell regeneration and other aspects. Since exosomes contain a large amount of genetic information and bioactive substances, they can reach other cells and tissues through the circulatory system and produce remote regulatory effects. Exosomes have a wide range of industrial application value.

[0003] Exosomes play an important role in the pathological process of neuropsychiatric diseases. Guo Lin's team from Xuzhou Medical University published an article in Brain Behav Immun on how plasma exosomes from patients with Major Depressive Disorder (MDD) can alleviate LPS-induced depressive-like behaviors, and explained that its antidepressant mechanism is dependent on the Sigma-1 receptor on exosomes, thus providing a new targeted treatment method for MDD patients that relies on the Sigma-1 receptor on exosomes (Yun Wang, Ce Gao, et al. Tianyu Gao Plasma exosomes from depression ameliorate inflammation-induced depressive-like behaviors via sigma-1 receptor delivery. Brain Behav Immun 2021; 94: 225-234.)

[0004] There are many methods for preparing exosomes, such as ultracentrifugation, filtration centrifugation, density gradient centrifugation, immunomagnetic bead method, microfluidic separation method, and size exclusion chromatography. Among them, the immunomagnetic bead method or latex separation method has mild conditions, fast speed, low energy consumption, and low equipment requirements, and is deeply loved by companies. However, the core of the immunomagnetic bead or latex process is the type of selected antigen and the quality of the antibody. The core reagents of various products on the market are purchased from antibody companies, and the quality is uneven.

[0005] CD9 is a 24-27kDa membrane protein belonging to the tetraspanin family. CD9 is expressed on the surface of multiple cells, including most immune cells, such as B cells, CD4+T cells, CD8+T cells, natural killer cells, granulocytes, monocytes, macrophages, immature and mature dendritic cells, and endothelial cells. CD9 is also highly expressed in exosomes. The present invention has conducted an in-depth study on the characteristics of CD9, one of the exosome membrane proteins, and based on this, prepared a more efficient CD9 polyclonal antibody-coated latex microsphere for exosome preparation. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a polypeptide sequence and a method for preparing exosomes for treating depression, wherein the polypeptide sequence can be used to efficiently separate exosomes.

[0007] The first object of the present invention is to provide a polypeptide sequence, which consists of the following four polypeptides:

[0008] (1)NCCGLAGGVEQFISDICPK;

[0009] (2)NCCGLAGGVEQFISDICPKKDVLETFTVKSC;

[0010] (3)PKKDVLETFTVKS:

[0011] (4)GLAGGVEQFISDICPK.

[0012] The second object of the present invention is to provide a polyclonal antibody mixture, wherein the above polypeptides are used to immunize animals respectively to prepare four polyclonal antibodies, and the four polyclonal antibodies are mixed to obtain a polyclonal antibody mixture.

[0013] Preferably, the animal is a rabbit.

[0014] Preferably, in the polyclonal antibody mixture, the volume ratio of the four polyclonal antibodies is 1:1:1:1.

[0015] The third object of the present invention is to provide a method for preparing latex microspheres, wherein the above-mentioned polyclonal antibody mixture is coupled to the surface of latex microspheres to obtain latex microspheres.

[0016] Preferably, the polyclonal antibody mixture is coupled to the surface of latex microspheres using the EDC method to obtain latex microspheres.

[0017] The fourth object of the present invention is to provide a latex microsphere for isolating exosomes for treating depression, which is prepared by the above method.

[0018] A fifth object of the present invention is to provide the use of the latex microspheres described above in extracting and isolating exosomes; preferably, the exosomes are exosomes used to isolate and treat depression.

[0019] Preferably, the application is as follows: the cells are broken and then centrifuged to collect the supernatant, the latex microspheres are added for extraction, and the precipitate is collected after centrifugation to obtain exosomes;

[0020] Preferably, the method further comprises the step of washing the precipitate with PBS.

[0021] The sixth object of the present invention is to provide a method for preparing exosomes for treating depression, wherein the cells are broken and then centrifuged to collect the supernatant, the latex microspheres are added for extraction, and the precipitate is collected after centrifugation to obtain exosomes;

[0022] Preferably, the method further comprises the step of washing the precipitate with PBS.

[0023] The technical effects of the present invention are:

[0024] Compared with ultracentrifugation and chip methods, the latex method for separating exosomes has lower requirements for instruments and equipment, and the method is mild. The separated exosomes have good integrity and high purity. However, the core reagents prepared by latex microspheres have varying antibody levels, which seriously affect the exosome yield and quality. The present invention achieves good exosome separation effects by optimizing polypeptides to prepare latex microsphere core reagents. The technical core of the invention lies in polypeptide design and optimization. Experiments have shown that strict design of new polypeptides is the key to success.

[0025] Through the separation and purification steps of high performance liquid chromatography, a polypeptide with a purity higher than 90% can be obtained, which meets the requirements of the immunoassay. The ELISA detection method proves that the antibody has high sensitivity and titer.

[0026] The latex microspheres were coated with a mixture of anti-CD9 polyclonal antibodies through covalent linkage, and the reliability and accuracy of the exosome isolation results were ensured through experimental testing and verification.

[0027] Exosomes have been shown to play a role in alleviating depression. The present invention obtains polyclonal antibodies with high affinity for cell exosomes, and uses them as core raw materials to make latex microspheres for exosome separation and preparation. By optimizing the affinity of the coated antibodies of the latex microspheres, the efficiency of exosome separation and preparation is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1This is a hydrophilicity analysis of CD9 (Human) protein.

[0030] Figure 2 The figure is a schematic diagram of the activation of the polypeptide of the present invention and the Fmoc-AA prepared in the early stage by reacting with DCC and HOBT.

[0031] Figure 3 The diagram is a schematic diagram of the reaction of connecting the polypeptide of the present invention and the activated amino acid prepared therefrom to resin.

[0032] Figure 4 This is a graph showing the ELISA test results of the anti-CD9 mixed polyclonal antibody of the present invention.

[0033] Figure 5 This is a target protein recovery rate detection diagram of the latex microspheres of the present invention.

[0034] Figure 6 It is a comparison chart of the detection effect of separating exosomes of the latex microspheres of the present invention. DETAILED DESCRIPTION

[0035] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0037] The present invention prepares four amino acid fragments by an in vitro synthesis method, induces the production of four polyclonal antibodies with high affinity against CD9, and then prepares a mixture of the four polyclonal antibodies, and covalently connects them to latex microspheres. The latex microspheres can effectively bind to exosomes, and the exosomes can be separated by short-term centrifugation, thereby avoiding the stringent requirements of ultracentrifugation on machinery and equipment and reducing production costs. At the same time, the polyclonal antibody mixture prepared by optimizing the amino acid sequence and the adjuvant can bind to the CD9 protein from all angles, thereby improving the affinity of the antibody to the exosome surface marker CD9, thereby improving the level of exosome separation and purification.

[0038] Example 1 Preparation of polypeptide

[0039] The complete amino acid sequence of 1CD9 (Human) is as follows (SEQ ID NO.1 in the sequence list):

[0040] MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHK DEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV

[0041] 2 The protean in DNAstar software was used to analyze the characteristics of CD9 protein (Human). Including secondary structures such as α helix, β fold, β turn, random coil; as well as hydrophilicity, flexibility, antigenicity, and surface accessibility. B cell epitopes tend to be in the β turn and random coil regions with high hydrophilicity, flexibility, antigenicity, and surface accessibility scores. Table 1 shows the α helix sites of CD9 protein.

[0042] Table 1 Alpha helical sites of CD9 (Human) protein

[0043] α-helical site 1 5-37 2 41-45 3 55-82 4 86-111 5 114-133 6 137-139 7 140-150 8 161-166 9 173-175 10 181-190 11 193-222

[0044] 3Analyze the hydrophilicity of CD9 (Human) protein. The results are as follows Figure 1 shown.

[0045] Figure 1 This is a hydrophilicity analysis of CD9 (Human) protein.

[0046] 4 Analysis of the amino acid physicochemical properties of CD9 (Human) protein. The results are shown in Table 2.

[0047] Table 2 Analysis of amino acid physicochemical properties of CD9 (Human) protein

[0048]

[0049] 5 In summary, 384 different antigen combinations were obtained according to the design calculations, as shown in Table 3.

[0050] Table 3 384 different antigen combinations

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] 6 Based on experience, the bold and underlined sequences were selected and adjacent amino acids were appropriately added or deleted for experiment. Finally, the following four amino acid sequences were selected for polyclonal antibody preparation:

[0061] Amino acid sequence 1 (SEQ ID NO.2 in the sequence listing):

[0062] NCCGLAGGVEQFISDICPK.

[0063] Among them, the 2nd, 3rd and 17th positions are all cysteine.

[0064] Amino acid sequence 2 (SEQ ID NO.3 in the sequence listing):

[0065] NCCGLAGGVEQFISDICPKKDVLETFTVKSC.

[0066] Among them, the 2nd, 3rd and 17th positions are all cysteine.

[0067] Amino acid sequence three (SEQ ID NO.4 in the sequence list):

[0068] PKKDVLETFTVKS.

[0069] Among them, the 2nd, 3rd and 12th positions are all lysine.

[0070] Amino acid sequence four (SEQ ID NO.5 in the sequence listing):

[0071] GLAGGVEQFISDICPK.

[0072] Among them, the 4th and 5th positions are glycine, and their carbon ends are linked to valine and glutamic acid.

[0073] 7. The amino acid sequence 1 is prepared by a method in the prior art, comprising the following steps:

[0074] Step 1. Experimental material preparation: prepare P-hydroxymethylphenoxymethyl polyvinyl resin, Wang resin, Fmoc-AA, NMP nitrogen methyl pyrrolidone, DCM dichloromethane, MeoH methanol, Piperidine piperidine, DMAP dimethylaminopyridine, HOBT hydroxybenzotriazole, DCCN, N'-dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1,2-ethanedithiol, thioanisole, crystalline phenol, acetonitrile;

[0075] Step 2. Preparation of experimental instruments: prepare automatic peptide synthesizer, rotary evaporator, high performance liquid chromatograph, freeze dryer, vacuum circulating water pump, centrifuge;

[0076] Step 3. Activation of Fmoc-AA: Fmoc-AA was activated by reaction with DCC and HOBT;

[0077] Step 4. Activate the amino acid to connect the resin: react the activated amino acid with P-hydroxymethylphenoxymethyl polyvinyl resin under DMAP conditions to obtain a resin connected with the amino acid;

[0078] Step 5. Removal of the Fmoc protecting group connected to the amino acid resin: under the action of piperidine, the Fmoc protecting group connected to the amino acid resin is removed;

[0079] Step 6. Amino acid activation: Activate the amino acid by reacting with DCC and HOBT;

[0080] Step 7. Coupling of activated carboxyl components: coupling the resin obtained in step 4 with an excess of activated carboxyl components, and removing the amino protecting group of the activated amino component after coupling;

[0081] Step 8. Preparation of peptide resin: Repeat the above steps 3 to 7, and the activated carboxyl components coupled each time are NCCGLAGGVEQFISDICPK from left to right to obtain the peptide resin;

[0082] Step 9. Separation of polypeptides: react trifluoroacetic acid with a scavenger mixed with 1,2-ethanedithiol, thioanisole and water to separate the polypeptide fragments from the peptide resin; filter out the resin after the reaction, and remove the scavenger by vacuum distillation; dissolve the polypeptide fragment in water, and extract with ether to obtain a crude polypeptide compound. The obtained polypeptide fragment is named ECD9151.

[0083] Step 10. Separation, purification and analysis of polypeptides: The crude polypeptide compound was separated and purified by high performance liquid chromatography. Specifically, the operating conditions were as follows: chromatographic column: C1825×250mm, chromatograph: Waters, mobile phase: A-volume percentage 0.1% TFA, H2O, B-volume percentage 0.1% TFA in volume percentage 60% acetonitrile, detection wavelength: 214nm, flow rate: 10ml / min, and the initial elution concentration was set at a volume concentration ratio of solution A:B of 2:8. In half an hour, the concentration ratio of solution A:B slowly increased to 4:6. By continuously increasing the concentration of solution B linearly, a purified polypeptide was obtained, and the purity was qualified with a purity of >90%.

[0084] Furthermore, the separated and purified CD9 polypeptide was subjected to high performance liquid chromatography analysis, and the analysis conditions were as follows: chromatographic column: C184.6×150 mm, mobile phase: A-0.1% TFA by volume, H2O, B-0.1% TFA by volume, acetonitrile, detection wavelength: 214 nm, flow rate: 1 ml / min, elution gradient from 0-60% B.

[0085] Figure 2 The figure is a schematic diagram of the activation of the polypeptide of the present invention and the Fmoc-AA prepared in the early stage by reacting with DCC and HOBT.

[0086] Figure 3 The diagram is a schematic diagram of the reaction of connecting the polypeptide of the present invention and the activated amino acid prepared therefrom to resin.

[0087] 8 The same method as step 7 was used to prepare amino acid sequence 2. The coupled activated carboxyl components were as follows from left to right: NCCGLAGGVEQFISDICPKKDVLETFTVKSC. The obtained polypeptide fragment was named ECD9152 and was qualified if the purity was >90%.

[0088] The same method as step 7 was used to prepare amino acid sequence three. The coupled activated carboxyl components were from left to right: PKKDVLETFTVKS. The obtained polypeptide fragment was named ECD9153 and was qualified if the purity was >90%.

[0089] The same method as step 7 was used to prepare amino acid sequence 4. The coupled activated carboxyl components were from left to right: GLAGGVEQFISDICPK. The obtained polypeptide fragment was named ECD9154 and was qualified if the purity was >90%.

[0090] Example 2 Preparation of polyclonal antibodies

[0091] 1. Reagents and peptides:

[0092] Freund's complete adjuvant: purchased from Merck, product number: F5881.

[0093] Freund's incomplete adjuvant, improved: purchased from Merck, catalog number: 344291.

[0094] Control peptide fragment: GGGGSGGGGSGGGGS was used as a control.

[0095] 2. Preparation of polyclonal antibodies

[0096] First, the polypeptide fragment ECD9151 prepared in Example 1 was coupled with the carrier protein KLH (hemocyanin or keyhole limpet hemocyanin), and the coupling agent was 3-maleimidobenzoic acid succinimidyl ester (MBS, CAS: 58626-38-3). The coupling method was referred to the MBS specification.

[0097] Then the coupled polypeptide fragments are fully emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant, respectively, to obtain fully emulsified complete adjuvant + polypeptide antigen, and fully emulsified incomplete adjuvant + polypeptide antigen, respectively.

[0098] Finally, the New Zealand white rabbits used in the experiment were first subjected to basic immunization (complete adjuvant + fully emulsified polypeptide antigen), and then multiple booster immunizations (incomplete adjuvant + fully emulsified polypeptide antigen) were performed on the back and foot pads at weeks 3, 5, and 7. Blood was collected from the ear vein on the 7th day after immunization, and the serum was separated. The antibody titer of CD9 was identified by ELISA. When the CD9 antibody concentration in the serum reached a peak, the rabbit with the best test data was taken, blood was collected from the carotid artery, and the serum was separated. The anti-CD9 rabbit polyclonal antibody ECD9151 was obtained by Protein A / G affinity purification.

[0099] The polypeptide fragments ECD9152, ECD9153 and ECD9154 in Example 1 were prepared according to the above method to obtain the corresponding anti-CD9 rabbit polyclonal antibodies ECD9152, ECD9153 and ECD9154.

[0100] Then, the four polyclonal antibodies were mixed in equal volumes to obtain a mixed polyclonal antibody.

[0101] The method for immunizing New Zealand white rabbits with the control peptide fragment was the same as above.

[0102] 3. Sensitivity of polyclonal antibodies

[0103] The sensitivity, i.e., titer, of the antibody was tested by ELISA. Figure 4 .

[0104] Figure 4 This is a graph showing the ELISA test results of the anti-CD9 mixed polyclonal antibody of the present invention.

[0105] The test results showed that the titer of CD9 antibody reached 1:30,000, and the sensitivity of the antibody was very high. The titer of the control anti-CD9 rabbit polyclonal antibody obtained by immunization with the control peptide fragment was lower than 1:5000, proving that qualified antibodies had been obtained.

[0106] The results showed that the anti-CD9 rabbit polyclonal antibody prepared by the present invention can bind well to CD9 and the titer reaches 1:30,000.

[0107] Example 3 Preparation of latex microspheres

[0108] 1 Prepare the following buffer:

[0109] Reaction buffer: 50 mM MES, pH 5.0 (chilled on ice).

[0110] Blocking buffer: 50 mM HEPES, pH 7.4, 1% (w / v) BSA.

[0111] Storage buffer: 50 mM HEPES, pH 7.4, 1% (w / v) BSA, 0.05% (w / v) 300.

[0112] 2. The protein (i.e., the polyclonal antibody mixture prepared in Example 2) was coupled to the surface of the latex microspheres using the EDC method to obtain latex microspheres.

[0113] According to the solid content of the product, 10 mg of latex microspheres (Suzhou Weidu Biotechnology Co., Ltd. Product No. PS02) were taken out and added to a 2 mL round-bottom centrifuge tube, and the microspheres were diluted with reaction buffer to a solid content of 1% w / v. The latex microsphere dispersion was thoroughly mixed and balanced to room temperature. 1 mg of protein (i.e., the polyclonal antibody mixture prepared in Example 2) was added to the above reaction solution, and it was thoroughly inverted and mixed, and then placed on a mixer and incubated at 25°C for 30 min; 10-20 mg of EDC solid was weighed and dissolved in reaction buffer precooled to 4°C to make an EDC concentration of 10 mg / mL, and the EDC solution was added to the above round-bottom centrifuge tube, and the protein was thoroughly mixed. The amount of EDC added was 1.5 times the molar ratio of the total amount of carboxyl groups, and the reaction was carried out on a mixer at 25°C for 3 h.

[0114] After the reaction is completed, centrifuge at 5000×g for 15 minutes, remove the supernatant, add 5mL of reaction buffer, use a vortex mixer to break up the precipitated microspheres and resuspend them, and then remove the supernatant. Repeat this step of washing for a total of 3 times, add 5mL of blocking buffer to the precipitate, use a vortex mixer to initially break up the precipitated microspheres, then place the tube on ice, insert the ultrasonic disruptor probe, use a power of about 200W for ultrasonic treatment for 10-15 minutes, each treatment for 5s, cool for 5s, place on a mixer after treatment, block the reaction at 25°C overnight, centrifuge overnight, remove the supernatant, then add 5mL of preservation solution, use an ultrasonic probe to completely break up, and store at 4°C for later use.

[0115] 3. Detection of the effect of latex microspheres

[0116] The latex microspheres prepared by the present invention were used as the experimental group, and three groups of control products were set up for control testing. The three groups of control products were all commercially available products, namely control product 1, control product 2 and control product 3.

[0117] Take 20ml of the cell supernatant containing exosomes and centrifuge at low speed, centrifuge at 500g for 15 minutes to remove cell debris and large particles, collect the supernatant and transfer it to a new centrifuge tube; add 1.5ml of latex microspheres prepared in step 2, mix well, let stand for 30 minutes, centrifuge at 5000G for 10-20 minutes to precipitate the exosomes bound to the latex, and collect the exosome precipitate; wash the exosome precipitate with PBS at pH 7.2 to remove contaminants such as cell residues and serum proteins, and add enough washing buffer to gently suspend the exosome precipitate and wash it multiple times to improve the purity of the sample. Add protease inhibitors to the exosome-containing precipitate, and quantify the exosome protein obtained by the BCA method. Use Western-Blot to identify the exosome characterization protein CD9. Use 10μL of CD9 protein marker with a concentration of 1mg / ml as a calibrator, and determine the relative amount of CD9 protein in the extract by grayscale scanning.

[0118] At the same time, another exosome extraction sample was taken, and the particle size distribution and concentration of the exosomes were analyzed by the microfluidic chip method: the above resuspended exosome sample was diluted according to the operating instructions so that the number of particles after the sample dilution was within the optimal range of 5,000-15,000, and the exosome concentration and particle size distribution were measured using a microfluidic analysis machine.

[0119] Figure 5 This is a target protein recovery rate detection diagram of the latex microspheres of the present invention.

[0120] Figure 6 It is a comparison chart of the detection effect of separating exosomes of the latex microspheres of the present invention.

[0121] from Figure 5and Figure 6 It can be seen that the latex microspheres of the present invention can effectively extract exosomes.

[0122] from Figure 6 It can also be seen that the diameter of the exosomes is between 40 and 150 nm, and the isolated exosomes are highly intact.

[0123] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide composition, characterized in that: The polypeptide composition consists of the following four polypeptides: (1) NCCGLAGGVEQFISDICPK; (2)NCCGLAGGVEQFISDICPKKDVLETFTVKSC; (3) PKKDVLETFTVKS: (4) GLAGGVEQFISDICPK.

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