Brucella CTL antigen epitope peptide and application thereof

Abstract

The invention discloses a Brucella CTL (cytotoxic T lymphocyte) antigen epitope peptide and application thereof, the Brucella CTL antigen epitope peptide is any one or more of the following epitope polypeptides, and the amino acid sequences of the epitope polypeptides are shown as SEQ ID NO.1-3; according to the present invention, the CTL immune response is accurately activated, the immune defense requirement of Brucella intracellular infection is met, the core component is provided for the novel Brucella subunit vaccine, and the risks of virulence reversion, diagnosis interference and the like of the traditional attenuated live vaccine are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antigen epitope polypeptides, and particularly relates to a Brucella CTL antigen epitope peptide. BACKGROUND

[0002] Brucellosis (Brucellosis) is a zoonosis caused by Brucella infection, mainly prevalent in cattle, sheep and other livestock, with the rapid development of China's animal husbandry in recent years, the flow and transaction of livestock become more frequent, which leads to the prevalence of Brucella in livestock showing an upward trend. Brucella has strong transmission ability, which can cause animal abortion and even death, and seriously affects the health and reproductive performance of animals. Vaccine immunization is an important measure to prevent Brucella infection, and the currently marketed Brucella vaccine is mainly attenuated live vaccine, although it plays a certain role in controlling Brucellosis, but there are still problems such as "virulence reversion", interference with serological diagnosis, and infection risk to humans. Therefore, it is urgent to develop a new type of vaccine with high safety and good immunization effect to cope with the limitations of the current vaccine and meet the needs of the public health field.

[0003] As an intracellular parasite, the immune response of Brucella mainly depends on cellular immunity, especially the major histocompatibility complex I (MHCI) antigen epitope presentation, which plays a key role in activating cytotoxic T cell immune response (CTL) reaction. Therefore, it is urgent to explore effective epitopes for CTL immune response, which provides a new possibility and direction for the development of new subunit vaccines to prevent Brucella infection. Therefore, a Brucella CTL antigen epitope peptide and its application are proposed. SUMMARY

[0004] The purpose of the present application is to solve the problems in the background art, and a Brucella CTL antigen epitope peptide and its application are proposed.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: A Brucella CTL antigen epitope peptide is any one or more of the following epitope polypeptides, and the amino acid sequence of the epitope polypeptide is as shown in SEQ ID NO. 1-3.

[0006] A coding gene of a Brucella CTL antigen epitope peptide.

[0007] A biological material of a Brucella CTL antigen epitope peptide coding gene, wherein the biological material is a recombinant expression vector, an expression cassette, a recombinant bacteria or a host cell.

[0008] A medicine containing the Brucella CTL antigen epitope peptide.

[0009] A polypeptide vaccine contains the Brucella CTL epitope peptide.

[0010] Further, the polypeptide vaccine also contains an adjuvant, which includes chitosan, carrier protein.

[0011] In one aspect, the Brucella CTL epitope peptide or its encoding gene or the biological material is used for preparing a vaccine for preventing Brucella infection.

[0012] In another aspect, the Brucella CTL epitope peptide or its encoding gene or the biological material is used for preparing a drug for Brucella infection.

[0013] In another aspect, the Brucella CTL epitope peptide or its encoding gene or the biological material is used for preparing a reagent or kit for detecting Brucella.

[0014] Compared with the prior art, the present application provides a Brucella CTL epitope peptide, which has the following beneficial effects: The present application precisely activates CTL immune response, meets the immune defense requirement of intracellular infection of Brucella, provides a core component for a new type of Brucella subunit vaccine, and avoids the risks of traditional attenuated live vaccine virulence reversion, interference with diagnosis, etc. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 .HLA-A*0201, beta 2m protein expression diagram; Wherein, M: protein standard molecular weight; 1: HLA-A*0201 uninduced; 2: HLA-A*0201 inclusion body; 3: HLA-A*0201 supernatant; 4: mouse-beta 2m uninduced; 5: mouse-beta 2m supernatant; 6: mouse-beta 2m inclusion body; the arrow points to the target protein; Figure 2 .Mouse spleen index and bacterial load diagram; Figure 3 Epitope peptide assisted HLA-A*0201 complex in vitro renaturation result diagram; Wherein, Figure 3 A.pHLA-A*0201 molecular sieve chromatography result; B.pHLA-A*0201 ion exchange result; Figure 4 Screening peptide stability identification diagram; Figure 5 Elispot method screening polypeptide stimulating IFN-gamma secretion result diagram. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application.

[0017] Example 1 Identification of polypeptide binding motif First, the HLA-A*0201 (GenBank: FN806801.1) and hβ2m (GenBank: NM_004048.4) gene sequences were synthesized, and the HLA-A*0201 and hβ2m gene sequences were respectively connected with the expression vector pET28a to successfully construct the pET28a-HLA*0201 and pET28a-hβ2m recombinant plasmids, which were transformed into BL21 competent cells for protein small amount induction expression analysis. The results showed that the heavy chain and light chain protein expression bands were correct and existed in the inclusion body. The recombinant protein was induced for expression and the inclusion body was extracted. After ultrasonic bacterial liquid was centrifuged, the bacterial fragments were gently pushed away using a glass rod, and the exposed white and dense bacterial blocks were the inclusion bodies. After the inclusion bodies were resuspended with inclusion body purification buffer and centrifuged, they were repeated 4 times. After the inclusion bodies were dissolved with 30 mg / ml guanidine hydrochloride, the protein HLA-A*0201, β2m and random nonapeptide were co-renatured, and the renaturation liquid was added in a molar ratio of 1:3:1. After the renaturation was completed, the renaturation liquid was concentrated to 50 ml using a 30KD concentration cup, 3 volumes of molecular sieve solution were added, and the concentration was continued to 1-2 ml. After filtration with a 0.22 μm filter, it was ready for use. After the Supredex20010 / 300 gel chromatography column was equilibrated with the molecular sieve buffer, the protein was injected into the protein liquid chromatograph AKTA sample ring, and different molecular weight proteins were collected for SDS-PAGE analysis. In order to further obtain purer human MHC I complex, the protein peak of the target protein complex correctly identified by SDS-PAGE was collected, and further purified by anion exchange column. The elution peak was collected for SDS-PAGE analysis. The results showed that the human MHC I heavy chain, light chain and random nonapeptide formed a complex and tightly combined together during the renaturation process. The purified human MHC I complex was concentrated to 200 μl by 10KD ultrafiltration tube, collected into a new 1.5 ml EP tube, 3 volumes of 10% acetic acid were added, and denaturation was performed at 65° for 15 min. The polypeptide was further eluted using a 3KD ultrafiltration tube. Finally, the treated polypeptide was desalted, dried and ready for use.

[0018] Example 2 Construction of animal infection model In this example, B-HLA-A2.1 mice were infected with the live attenuated vaccine strain Rev.1 of ovine Brucella abortus. In this example, 18 mice were used, 3 mice in each group, a total of 6 groups, and the mice were injected intraperitoneally with 1×10 5Rev.1 strain was administered at a dose of CFU / 100 μl. Spleens were collected from mice under sterile conditions at 1, 5, 14, 28, 50, and 90 days post-immunization, and mouse body weight and spleen weight were measured. The spleen index was calculated. 0.1 g of spleen cell suspension was prepared, plated, and colony counts were performed. The remaining portion was stored at -80°C for later use. Results showed that the spleen index of mice significantly increased after immunization, and the obtained colony counts reached a quantifiable and stable titer level. These data clearly indicate that a significant immune response was generated in mice after Brucella infection. The mouse model has been successfully established, providing a reliable experimental platform for further research on the immune mechanism of brucellosis and vaccine development.

[0019] Example 3: Isolation and Identification of Mouse Lymphocytes In this example, 4-5 ml of mouse lymphocyte separation medium was placed in a 35 mm culture dish. The mouse spleen obtained in Example 2 was placed on a nylon mesh or cell sieve and immersed in the separation medium. The spleen was then ground in the separation medium using a syringe plunger. The separation medium containing the suspended spleen cells was transferred to a 15 ml centrifuge tube, and RPMI 1640 medium was slowly added (keeping the liquid level clearly defined). The tube was centrifuged at 800 g for 30 min at room temperature with a slow acceleration and deceleration rate. The lymphocyte layer was collected, 10 ml of RPMI 1640 medium was added, the tube was inverted and washed, and the tube was centrifuged at 250 g for 10 min at room temperature. The cells were then collected.

[0020] Example 4: Peptide Elution The MAE (mildacidelution) buffer used in this example consists of 131 mM citric acid, 66 mM Na2HPO4, 150 mM NaCl, 1 M maprotinin, and 25 mM iodoacetamide, adjusted to pH 3 with NaOH. The lymphocytes isolated in Example 3 were resuspended in pre-chilled PBS and centrifuged at 250 g for 10 min, repeated three times. 4 ml of MAE was added, and the lymphocytes were repeatedly pipetted and centrifuged at 258 g-300 g for 5 min to remove cells. The supernatant was transferred to a new centrifuge tube and centrifuged at 339 g-350 g for 10 min. The supernatant was then transferred to another new centrifuge tube and centrifuged at 3345 g-3400 g for 15 min. The supernatant was collected and centrifuged at 20000 g for 1 h at 4°C. The supernatant was then stored at -80°C for later use. Peptides eluted with a weak acid gradient were desalted using 0.5% IFA (trifluoroacetate), 80% ACN (acetonitrile), and 10% IFA, and the peptides were then dried before being loaded onto the instrument.

[0021] Example 5: Mass Spectrometry Identification In this embodiment, the peptides obtained in Examples 1 and 4 were coupled to an OritrapFusionLumosMS (ThermoFisher Scientific) system via an electrospray interface using nanocapillaries, and the peptides were resolved by capillary chromatography. MHC peptides were eluted using a gradient of 0.1% formic acid and 6%–36% acetonitrile at a flow rate of 0.3 μl / min. Direct elution (DDA) was performed using the following parameters: MS1 resolution was set to 120,000, normalized AGC was 200%, dynamic exclusion time was set to 30 s, and a mass tolerance of ±10 ppm was allowed. Collision energies of 25%, 30%, and 35% were used for HCD. For MS2, the resolution was set to 50,000, the normalized AGC target was 200%, and the maximum injection time was 86 ms. The LC-MS results were identified and quantified using Mascot, and a library search analysis was performed.

[0022] Example 6: Analysis of Peptide Binding Motifs In this embodiment, the binding peptide motif of the MHCI molecule depends on the fit between the anchor residues of the peptide and the pockets of the MHCI peptide binding groove. The peptide binding groove of MHCI is determined by the size, acidity / basicity, and hydrophobicity of the amino acid side chains. Therefore, some pockets of the peptide binding groove have strong restrictions on the bound amino acids; these amino acids are the main anchor sites for the binding peptide. Studies have shown that the main anchor sites for human MHCI peptides are P2 and PC. By comparing the main anchor sites of the peptide binding motifs obtained in Examples 1 and 4, the preference of MHCI peptide binding motifs can be analyzed, and the reliability of the peptide binding motif obtained in Example 4 can be verified. The results show that the anchor residues at the P2 position of the peptide in Example 1 are L, M, I, and V, and the anchor residues at the Pc position are V, L, I, and A. Selecting peptide binding motifs conforming to Example 1 and performing in vitro refolding showed that peptides conforming to the binding motifs could fold correctly.

[0023] The peptides eluted directly in mice conformed to the binding motifs of random peptides bound in vitro by MHCI, further demonstrating the reliability of the screened peptides. This proves that the present invention has innovatively developed a novel method for identifying Brucella CTL epitopes, and the screened peptides hold promise as key components for developing novel subunit vaccines.

[0024] In summary, this invention has developed a novel method for identifying Brucella CTL epitopes to compensate for the shortcomings of bioinformatics prediction and to provide support for the development of novel subunit vaccines to prevent Brucella infection.

[0025] Example 7: Identification of Epitope Peptide Stability (a) Cell plating: Option A: Use a 6-well plate, add 5×10 to each well. 5Mix the target cells thoroughly and incubate at 37°C for 8 hours.

[0026] Option B: First, incubate at 37℃ for 8 hours, then transfer to 26℃ for 12 hours.

[0027] (ii) Peptide dissolution: Each tube of synthesized peptide weighs 4.75 mg. Add 50 μL DMSO to completely dissolve the peptide, and then add 900 μL DMEM to dilute it (the diluted peptide concentration is 5 mg / mL). Each peptide is tested in three replicates.

[0028] (III) Peptide Loading: Centrifuge at 1000 rpm for 3 min, discard the culture medium, resuspend in PBS, and wash 3 times. Adherent cells should also be washed three times with PBS to ensure a clean cell growth environment. After washing, add 2 mL of culture medium to each plate, add 100 μL of peptide to the corresponding well, to a final concentration of 50 μg / mL, and add 3 μg / mL of β2m solution to each well. Mix well.

[0029] Meanwhile, control groups were set up with CMVpp65495–503 as the positive peptide (NLVPMVATV, 50 μg / mL) and OVA257–264 as the negative peptide (SIINFKEL, 50 μg / mL), and no peptide and β2-m were added at 37°C.

[0030] Protocol A: Incubate at 26℃ for 2 hours, then transfer to 37℃ for normal incubation for 4 hours.

[0031] Option B: Incubate at 37℃ for 16 hours.

[0032] (2) Flow cytometry to detect cell affinity for epitopes ① Cell collection: Digest all cells and collect them in a 1.5 mL tube, centrifuge and discard the supernatant; ② Blocking: Add 200 μL of filtered 3% BSA blocking solution to each tube, resuspend the cells and mix well. Block for 20 min at room temperature. After blocking, add an appropriate amount of PBS, centrifuge and discard the supernatant. Repeat twice to wash the blocking solution clean.

[0033] ③ Antibody incubation: Under light-protected conditions, add 200 μL of PE-tagged anti-HLA-A2.1 antibody diluted with universal antibody dilution buffer (1:500) to each tube, mix gently, and incubate at 4°C for one hour. Mix once every 10 minutes to ensure that the cells are in full contact with the antibody. ④ Cell detection sample preparation: After centrifuging each tube of cells and discarding the supernatant, resuspend in PBS and retain 300 μL. Filter the cells into single cells using a nylon mesh to ensure that the collected cells do not clump together. ⑤ Detection: After setting the flow cytometry parameters using the control group cells, the samples were tested.

[0034] The results showed that the FI values ​​of peptides VV, GL, and AL were 4.51, 3.71, and 3.51, respectively, indicating that these three peptides were high-affinity peptides. TL and YQ were low-affinity peptides. (See attached table). Figure 4 Table 1 shows that 5 Brucella CTL epitope peptides were screened, of which 3 were high-affinity peptides (FI≥3.51) and 2 were low-affinity peptides. The high-affinity peptides are shown in SEQ ID NO.1-3.

[0035] Example 8 Enzyme-linked immunospot assay Reagent preparation: ① Incomplete RPMI 1640 medium ② Complete RPMI 1640 medium: 10% FBS + 1% antibiotics ③ Commercial PMA+Ion: Purchased from Dakowei (Cat: 2030421). The powder in the bottle was diluted with 500 μl of PBS to prepare the working solution (taking 100 μl of cell culture medium per well as an example, 10 μl of working solution was added to each well, i.e., the concentration was PMA 500 ng / ml + Ion 10 μg / mL, and it was used to treat 1×10 5 (Each cell can produce a distinct positive spot) ④ Sterile PBS ⑤ Peptide dilution buffer: The concentration of the peptide stock solution diluted with DMSO is 20 mg / mL. Take 2 μl of the stock solution and dilute it in 1000 μl of PBS, that is, dilute it 500 times. At this time, the concentration is 40 μg / mL. ⑥ PBS-0.5%FBS: 50ml PBS + 250μl FBS (used to dilute antibodies R4-6A2-biotin and Streptavidin-HRP) ⑦ Deionized water Preparation of plate A (under sterile conditions) 1. Assemble the required number of strip plates in the plate rack and wash 4 times with sterile PBS (200 μl / well). Place the remaining strip plates in a sealed bag and store at room temperature.

[0036] 2. Equilibrate the cell wells with 200 μL of complete RPMI 1640 medium. Incubate at 37°C for at least 30 min.

[0037] B. Culture cells in a plate (under sterile conditions). Use a pipette to draw culture medium from the plate, then add 100 μL of cell suspension (adjusted to a concentration of 1 × 10⁻⁶ with complete culture medium) to each well. 7 Cells / ml, take 200μl and add it to 800μl of complete culture medium, add 100μl of diluted cell solution to each well, so that each well has 2×10⁶ cells / ml.5 (cells) a. Positive control wells: Add 20 μl of PMA+Ion working solution to each well. b. Experimental wells: The working concentration of the peptide is 2 μg / mL. Add 6 μl of peptide dilution solution to each well. c. Add 20 μl of PBS to the negative well. 2. Cover the plate and wrap it with aluminum foil. Transfer it to a CO2 incubator and incubate for 18-48 hours. Do not move the plate during this period and take measures to prevent evaporation.

[0038] C. Spot detection (not requiring sterility) 1. Empty the cells from the strip plate and wash 5 times with 200 μl / well of PBS.

[0039] 2. Dilute the detection antibody R4-6A2-biotin to 1 μg / ml using PBS-0.5%FBS: 10 ml PBS-0.5%FBS + 10 μl R4-6A2-biotin (stock solution concentration is 1 mg / ml) Then, add 100 μL to each well, wrap with aluminum foil, and incubate at room temperature for 2 h; 3. After antibody incubation, discard the liquid in the plate and wash 5 times with 200 μl / well sterile PBS. 4. Dilute the Streptavidin-HRP antibody 1:1000 using PBS-0.5% FBS: 10 ml PBS-0.5% FBS + 10 μl Streptavidin-HRP. Then add 100 μL to each well, cover with aluminum foil, and incubate at room temperature for 1 h. 5. Then clean it following step 3 above; 6. After cleaning, add 100 μL / well of ready-to-use TMB substrate solution and develop the color in the dark until obvious spots appear; 7. After the color development is complete, stop the color development with 200 μl / well of deionized water and wash 5 times. 8. After cleaning, wrap the product in aluminum foil and place it in a dark place to air dry naturally, then send it to the company for spot counting.

[0040] Note: Positive spots can be seen in 2-20 minutes, while spots in the experimental group usually appear after the membrane dries.

[0041] The results showed that the three peptides VV, GL, and AL could induce a strong CTL immune response, providing a basis for further experiments on epitope vaccines in animals. The results are shown in [see attached table]. Figure 5 .

[0042] Table 1. Comparison of polypeptide sequences and their fluorescence indices;

[0043] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Brucella CTL antigenic epitope peptide, characterized in that, It is any one or more of the following epitope peptides, the amino acid sequence of which is shown in SEQ ID NO. 1-3; the epitope peptide is a high-affinity epitope peptide, and the fluorescence index of the high-affinity epitope peptide binding to human MHCI molecules is greater than 3.

51.

2. The gene encoding a Brucella CTL antigenic epitope peptide according to claim 1.

3. A biological material containing a Brucella CTL antigenic epitope peptide encoding gene as described in claim 1, wherein the biological material is a recombinant expression vector, expression cassette, recombinant bacteria, or host cell.

4. A drug, characterized in that, It contains a Brucella CTL antigenic epitope peptide as described in claim 1.

5. A polypeptide vaccine, characterized in that, Contains the Brucella CTL antigenic epitope peptide as described in claim 1.

6. The polypeptide vaccine according to claim 5, characterized in that, The polypeptide vaccine also contains adjuvants, including chitosan and carrier proteins.

7. The use of the Brucella CTL antigenic epitope peptide of claim 1 or its encoding gene or the biological material of claim 3 in the preparation of a vaccine to prevent Brucella infection.

8. The use of the Brucella CTL antigenic epitope peptide of claim 1 or its encoding gene or the biomaterial of claim 3 in the preparation of a medicament for Brucella infection.

9. The use of the Brucella CTL antigenic epitope peptide of claim 1 or its encoding gene or the biological material of claim 3 in the preparation of reagents or kits for detecting Brucella.

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