Epitope chimeric beta amyloid protein as well as preparation method and application thereof

Abstract

The invention provides an epitope chimeric beta amyloid protein as well as application and a preparation method thereof. The epitope chimeric beta amyloid protein comprises A beta 1-42 and 1-15 site B cell epitopes of the A beta 1-42. According to the epitope chimeric beta amyloid protein disclosed by the invention, effective epitopes are fully exposed through flexible connection of A beta 1-42 and 1-15 B cell epitopes of the A beta 1-42, and relatively high humoral immune response is induced. Meanwhile, the invention also provides an efficient, soluble and active expression method of the epitope chimeric beta amyloid protein in escherichia coli.

Description

Field of the Invention

[0001] The present application belongs to the field of immunotechnology and Alzheimer's disease treatment. Specifically, the present application provides an epitope-chimeric amyloid β protein and a preparation method and use thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects people's physical health. Studies have shown that the main cause of AD is the excessive phosphorylation of Tau protein in the brain, which leads to the deposition of neurofibrillary tangles (NFTs) and β-amyloid (Aβ), which in turn leads to abnormal neuronal function. β-amyloid (Aβ) is a polypeptide containing 39-43 amino acids produced by the proteolysis of amyloid precursor protein (APP) by β- and γ-secretase. It can be produced by a variety of cells and circulates in the blood, cerebrospinal fluid, and brain interstitial fluid. Most of them are bound to chaperone protein molecules, and a few exist in a free state. The most common subtypes of Aβ in the human body are Aβ1-40 and Aβ1-42. In human cerebrospinal fluid and blood, the content levels of Aβ1-40 are 10 times and 1.5 times higher than those of Aβ1-42, respectively. Aβ1-42 has stronger fibrogenicity and neurotoxicity, and is more likely to aggregate, thus forming Aβ deposits such as senile plaques, which are involved in neurological diseases.

[0003] Studies have found that active immunization with Aβ1-42 and its subunit fragments as antigens and passive immunization with injection of Aβ antibodies can prevent and significantly reduce the deposition of senile plaques in the brains of AD model mice, and can significantly improve the behavioral and cognitive impairment of transgenic mice. At the same time, studies have found that the level of Aβ antibodies in AD patients is significantly lower than that in normal people, and the level of Aβ antibodies can be used for early warning of AD and as an indicator of the immune efficacy of Aβ subunit vaccines.

[0004] Since the B cell antigen epitopes of Aβ1-42 are mostly concentrated in 1-15aa, and the T cell antigen epitopes are in 15-42aa, the research on Aβ subunit vaccines is also focused on this. Currently, the mechanism of action of Lencanerumab, which has been launched for the treatment of AD, is to bind and clear the abnormal accumulation of Aβ, but it is not a drug that cures Alzheimer's disease, and can only alleviate and improve the symptoms of the disease. Therefore, there is still a great demand for effective vaccines for AD. Summary of the invention

[0005] Since Aβ is a polypeptide fragment of only more than 40 amino acids, and its B cell antigen epitope is only 15 amino acids, in order to fully expose the antigen epitope, the present invention links it with Aβ1-42 through flexible amino acids to form an epitope-chimeric Aβ protein. At the same time, in order to obtain soluble expression of the protein, a chaperone protein that can promote its expression, thioredoxin, was selected, and by optimizing the conditions for inducing expression, the expression amount of the target protein was higher and the activity was better.

[0006] The present invention connects Aβ1-42 and its main B cell epitopes 1-15 in series, and without changing its amino acid sequence, according to the E. coli expression system, through codon optimization, selects a suitable E. coli expression vector and induction expression conditions, so that it can be expressed efficiently and soluble in E. coli, so as to overcome the defects of low expression of recombinant protein and expression in inclusion bodies in the prior art. Immunizing mice with purified protein can produce stronger humoral immune response than simple Aβ1-42 immunization, providing a new idea for the development of ADβ-like amyloid protein vaccine.

[0007] On the one hand, the present application provides an epitope-chimeric amyloid β protein, characterized in that the epitope-chimeric amyloid β protein includes Aβ1-42 and B cell epitopes 1-15 of Aβ1-42.

[0008] Furthermore, the amino acid sequence of the Aβ1-42 is SEQ ID NO.2; the amino acid sequence of the B cell epitope at positions 1-15 of the Aβ1-42 is SEQ ID NO.3.

[0009] Furthermore, the epitope-chimeric amyloid-β protein also includes a flexible connecting portion.

[0010] Furthermore, the flexible linking portion consists of 1-5 repeating units of GGGGS.

[0011] Furthermore, the amino acid sequence of the flexible connecting part is SEQ ID NO.4.

[0012] Furthermore, the amino acid sequence of the epitope-chimeric amyloid β protein is SEQ ID NO.1.

[0013] On the other hand, the present application provides the use of the above-mentioned epitope-chimeric β-amyloid protein in the preparation of a drug for treating or preventing Alzheimer's disease.

[0014] On the other hand, the present application provides a plasmid comprising a nucleic acid encoding the above-mentioned epitope-chimeric amyloid-β protein.

[0015] According to the amino acid sequence of the protein and different hosts, those skilled in the art can design a suitable encoding nucleic acid according to known rules, such as codon preference.

[0016] Furthermore, the nucleotide sequence of the nucleic acid is SEQ ID NO.6.

[0017] On the other hand, the present application provides a method for preparing the above-mentioned epitope-chimeric β-amyloid protein, comprising: (1) culturing a host cell containing the above-mentioned plasmid; (2) expressing the epitope-chimeric β-amyloid protein; and (3) purifying the epitope-chimeric β-amyloid protein.

[0018] The host cell may be commercially available or homemade Escherichia coli, Bacillus subtilis, yeast, etc. The characteristics of these host cells and applicable plasmids and vectors are known to those skilled in the art.

[0019] Furthermore, in step (2), mM IPTG is used to induce the expression of the epitope chimeric amyloid β protein.

[0020] The present application connects the epitopes of Aβ1-42 and Aβ1-15 in series with a flexible linker in the middle. According to this design, the chimerization, full exposure and higher humoral immune response of the Aβ1-15 epitope can be achieved. At the same time, an E. coli expression vector and an induction purification method of an epitope-chimeric Aβ protein are provided to achieve its efficient, soluble and active expression in E. coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The SDS-PAGE detection results of Aβ42 and Aβ42 / 15, where R1 is Aβ42 and R2 is Aβ42 / 15.

[0022] Figure 2 These are the ELISA test results for the binding of Aβ42 and Aβ42 / 15 to commercial Aβ antibodies.

[0023] Figure 3 These are the results of humoral immune response detection in mice, where the Aβ42 group is mice immunized with Aβ42 protein, the Aβ42 / 15 group is mice immunized with Aβ42 / 15 protein, and the Ctrl group is mice immunized with carrier protein. DETAILED DESCRIPTION

[0024] Example 1 Synthesis of Aβ protein and its gene

[0025] According to the gene sequence of Aβ1-42, a (GGGGS)3 flexible amino acid linker was added, and then Aβ1-15 was connected in series to form a recombinant Aβ42 / 15 protein (72aa):

[0026] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAGGGGSGGGGSGGGGSDAEFRHDSGYEVHHQ (SEQ ID NO. 1);

[0027] Among them, Aβ1-42 is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO. 2) and Aβ1-15 is DAEFRHDSGYEVHHQ (SEQ ID NO. 3);

[0028] The flexible linker is GGGGSGGGGSGGGGS (SEQ ID NO. 4);

[0029] The flexible linker repeat unit is GGGGS (SEQ ID NO.5);

[0030] After E. coli codon optimization, the gene was synthesized and cloned into the Pet32A expression vector with thioredoxin and named Aβ42 / 15.

[0031] The optimized Aβ42 / 15 specific nucleotide sequence is (216nt):

[0032] GACGCAGAGTTCCGCCACGATTCCGGTTACGAAGTTCACCACCAGAAACTGGTATTCTTTGCTGAGGAC

[0033] GTGGGTAGCAACAAGGGTGCTATCATCGGCCTGATGGTCGGTGGCGTTGTTATCGCTGGCGGTGGTGGT

[0034] TCTGGTGGTGGTGGTTCCGGTGGCGGTGGTAGCGACGCTGAATTCCGCCACGACTCCGGTTATGAAGTGCACCACCAG (SEQ ID NO. 6);

[0035] At the same time, Aβ1-42 was also cloned into the pET32a vector for prokaryotic expression and named Aβ42.

[0036] Example 2 Expression and purification methods

[0037] The synthesized recombinant plasmids pET32a-Aβ42 / 15 and pET32a-Aβ42 were transformed into BL21 (DE3) competent cells according to conventional methods, and the transformed bacteria were spread on LB agar plates (containing 100 μg / mL ampicillin) and cultured at 37°C overnight. A single colony was picked and inoculated into 4 mL LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C, 230 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium (100 μg / mL ampicillin) and cultured at 37°C, 220 rpm for about 3 hours until the OD 600 The concentration of IPTG was 0.6, and the final concentrations were 0.1mM, 0.5mM, and 1mM respectively. The cells were collected under the following three conditions: 37°C, 180 rpm for 4 hours of induction; 25°C, 200 rpm for 8 hours of induction; 16°C, 180 rpm for 15 hours of induction.

[0038] Since the expressed recombinant proteins all carry histidine tags, we used ACHROM Base protein purification instrument and HisTrap TM HP chromatography column was used for preliminary crude purification. Buffer A was 10mM phosphate, 500mM NaCl, pH 8.0, and buffer B was 10mM phosphate, 500mM NaCl, 0.5M imidazole, pH 8.0. The column was balanced with buffer A, and then the fermented bacterial solution was centrifuged at 8000rpm for 5min. The precipitate was resuspended with liquid A, ultrasonically broken in an ice water bath for 20min, ultrasonicated for 3 seconds at intervals of 4 seconds, and centrifuged at 10000rpm for 30min. The supernatant was filtered with a 0.22 micron filter, loaded, and the column was washed with buffer A. Finally, the buffer B was used for gradient elution. The target protein collection peak was selected and dialyzed with 10mM phosphate pH 8.0 for liquid exchange, and then DEAE ion exchange chromatography was performed. The target protein peak was collected by gradient elution with different NaCl concentrations, and the purification was observed by SDS-PAGE protein gel electrophoresis. The protein concentration was determined using Thermo's ultra-micro spectrophotometer Nanodrop and stored at -20℃.

[0039] The optimal induction expression conditions for recombinant proteins were determined. Aβ42 and Aβ42 / 15 were both induced at 0.5 mM IPTG at 16°C and 180 rpm for 15 hours, and both achieved soluble expression, with the expression level of Aβ42 being 5.3 mg / L and the expression level of Aβ42 / 15 protein being 12.8 mg / L. After Ni column affinity chromatography and ion exchange chromatography, both proteins were able to obtain high purity.

[0040] Example 3 Identification of recombinant protein

[0041] Protein purity was determined by SDS-PAGE electrophoresis:

[0042] Protein sample pretreatment: add an equal volume of 2x SDS loading buffer to each sample, place in a boiling water bath for 10 minutes, centrifuge at 12000rpm for 3 minutes, and add the supernatant to the sample well. Preparation of SDS-PAGE electrophoresis gel: wash and dry the vertical electrophoresis glass plate, place it vertically on the gel rack, prepare the separation gel system as required, add the gap between the glass plates, and add anhydrous ethanol to seal after the gel surface reaches the required height; after solidifying at room temperature for 30min-1h, discard the sealing liquid on the upper layer of the gel, and dry it with absorbent paper, prepare the concentrated gel in proportion, insert a suitable comb, and solidify at room temperature for 30min-1h. SDS-PAGE electrophoresis: dilute the 5x glycine buffer to the working concentration, add it to the electrophoresis tank to the appropriate liquid level, and gently pull the comb out of the solidified gel. Add protein marker and 10μl treated protein sample to the sample well. Turn on the power supply, adjust the voltage to 90V constant voltage electrophoresis until the separation gel, and then change to 130V until bromophenol blue reaches the bottom of the gel. Staining and decolorization: Cut the gel from the glass plate, place it in Coomassie brilliant blue dye solution, shake and dye for 3 hours, and then decolorize with decolorizing solution until the bands are clear.

[0043] The results are as follows Figure 1 As shown, SDS-PAGE identification showed that the purity of the purified Aβ42 and Aβ42 / 15 proteins was high, greater than 85%, and the band positions were consistent with the expected sizes.

[0044] ELISA to identify the activity of recombinant protein

[0045] The purified recombinant Aβ42 and Aβ42 / 15 proteins were coated (1 ug / ml), and the reaction with the commercially available HyTest Aβ42 mouse monoclonal antibody (BAM7cc) was identified by ELISA. First, the recombinant protein was coated in the microplate (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate fixed to 1L pure water), the coating concentration was 1μg / mL, 50μl / well, 4℃ overnight; 3% sucrose 1% BSA blocking, 150μl per well, 37℃ for 2 hours, washed once with washing solution (PBST, PBS containing 0.05% Tween-20), and patted dry; the positive mouse monoclonal antibody was diluted with PBS according to the gradient of 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml, 1ng / ml, and added to 50μL of the microplate coated with the antigen. At the same time, other mouse monoclonal antibodies were used as negative controls and reacted at 37℃ for 30min. Shake out the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS), react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB color developing solution, develop at room temperature for 10 min, finally add stop solution to stop the reaction, and measure OD450nm value with a microplate reader.

[0046] The results are as follows Figure 2 As shown, indirect ELISA showed that both recombinant proteins Aβ42 and Aβ42 / 15 could specifically bind to commercial Aβ monoclonal antibodies, indicating that the protein had good biological activity and could be used for subsequent immune tests.

[0047] Example 3 Evaluation of the humoral immunity level induced by recombinant protein in mice

[0048] Mouse immunization:

[0049] The recombinant protein was identified to have biological activity, and its humoral immune response was evaluated by immunizing balb / c mice. The mice were randomly divided into 3 groups, 3 mice in each group, and immunized with recombinant proteins Aβ42, Aβ42 / 15, and control proteins expressed by pET32a vectors. Specifically, 3 female Balb / c mice aged 6 weeks were subcutaneously immunized at multiple points after being fully emulsified with an equal volume of Freund's complete adjuvant at a dose of 50ug / mouse. At 2 weeks and 4 weeks, the immunization was strengthened once again with Freund's incomplete adjuvant according to the above method. On the 7th day after the last immunization, the mouse serum was taken to detect the antibody titer. The immunization dose and immunization procedure of mice in different groups were the same.

[0050] Detection of Aβ1-15 specific antibodies in mouse serum

[0051] The plate was coated with commercially available BSA-Aβ1-15 peptide at a concentration of 1ug / ml, 50ul / well, overnight at 4℃; 3% sucrose, 1% gelatin blocking, 150μl per well, 37℃ for 2 hours, washed once with washing solution (PBST, PBS containing 0.05% Tween-20), and patted dry; the immune serum of the three groups of mice collected was diluted 4 times from 400 times, added 50μL to the microplate coated with antigen, and reacted at 37℃ for 30min. The liquid in the well was thrown out, the plate was washed 4 times with PBST washing solution, patted dry, 50μL / well of HRP-labeled goat anti-mouse (diluted with PBS at 1:5000) was added, reacted at 37℃ for 30min, and the plate was washed 4 times, patted dry, and 50μL / well of TMB colorimetric solution was added for color development at room temperature for 10min, and finally the stop solution was added to stop the reaction, and the OD450nm value was measured with an enzyme marker.

[0052] The results are as follows Figure 3As shown. In the levels of specific Aβ1-15 antibodies in balb / C mice immunized in different groups, both groups immunized with proteins containing the Aβ15 epitope produced specific antibodies against Aβ1-15, while the control carrier protein immunized mice group produced no specific antibodies. The results also showed that the antibody titer induced by epitope chimeric Aβ42 / 15 was 4 times higher than that of the Aβ42 group, indicating that the antigen of the chimeric epitope can induce a stronger level of humoral immunity, showing that the chimeric Aβ42 / 15 protein has better applicability.

Claims

1. An epitope-chimeric amyloid β protein, characterized in that: The epitope-chimeric amyloid β protein includes Aβ1-42 and B cell epitopes 1-15 of Aβ1-42.

2. The epitope-chimeric amyloid β protein according to claim 1, wherein the amino acid sequence of Aβ1-42 is SEQ ID NO.2; and the amino acid sequence of the B cell epitope at positions 1-15 of Aβ1-42 is SEQ ID NO.

3. 3 . The epitope-chimeric amyloid-β protein according to claim 2 , further comprising a flexible linker. The epitope-chimeric amyloid-β protein according to claim 3 , wherein the flexible linking portion consists of 1-5 repeating units of GGGGS. 5 . The epitope-chimeric amyloid β protein according to claim 4 , wherein the amino acid sequence of the flexible connecting portion is SEQ ID NO.

4. The epitope-chimeric amyloid β protein according to claim 5 , wherein the amino acid sequence of the epitope-chimeric amyloid β protein is SEQ ID NO.

1.

7. Use of the epitope-chimeric amyloid β protein according to any one of claims 1 to 6 in the preparation of a drug for treating or preventing Alzheimer's disease.

8. A plasmid, characterized in that The plasmid comprises a nucleic acid encoding the epitope-chimeric amyloid-β protein according to any one of claims 1 to 6.

9. The plasmid according to claim 8, wherein the nucleotide sequence of the encoding nucleic acid is SEQ ID NO.

6.

10. A method for preparing the epitope-chimeric amyloid β protein according to any one of claims 1 to 6, characterized in that: The method comprises: (1) culturing a host cell containing the above-mentioned plasmid; (2) expressing the epitope-chimeric amyloid-β protein; and (3) purifying the epitope-chimeric amyloid-β protein.

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