Epitope polypeptide of cryptosporidium parvum Gp900 protein and application

Abstract

The invention relates to an epitope polypeptide of cryptosporidium parvum Gp900 protein and application of the epitope polypeptide, the amino acid sequence of the epitope polypeptide is shown as SEQ ID NO.1, and the nucleotide sequence for coding the epitope polypeptide is shown as SEQ ID NO.2. Experiments prove that the epitope polypeptide has good in-vitro affinity with mouse H-2Db and has certain stability; after the epitope polypeptide is emulsified by an adjuvant to immunize a mouse, not only can CD8 + T cell immune response of the mouse be obviously stimulated, but also polypeptide specific CD4 + T cell response can be induced to be generated; compared with negative control, P is less than 0.01, and the IFN-gamma and the TNF-alpha generated by CD8 + T cells and CD4 + T cells induced by the Gp900 protein have extremely significant difference, so that the Gp900 protein and the related polypeptides thereof have corresponding biological immunology functions, have huge development and application potential in the field of cryptosporidium parvum specific immunotherapy, and have a broad application prospect in the field of cryptosporidium parvum specific immunotherapy. And a new direction is provided for preparing cryptosporidium parvum vaccines based on T cell epitopes.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to an epitope polypeptide of Cryptosporidium parvum Gp900 protein and an application thereof. Background Art

[0002] Cryptosporidium parvum belongs to the class Sporozoa of the phylum Apicomplexa. It was first reported and named by Tyzzer in 1912 and is an important zoonotic protozoan. Among the 31 reported zoonotic Cryptosporidium species, Cryptosporidium parvum can infect humans and most mammals, and has the widest host, mainly including humans, bovines, various rodents, camelids, equids, canines, non-human primates, marine mammals and fish. Like other apicomplexan parasites, in the process of Cryptosporidium parvum invading host cells, the complex protein mixture in the secretory organelles such as sporozoite rods, micronemes, and dense granules is released to the top of the sporozoite or the vesicle during the invasion of host cells, participating in the specific adhesion of Cryptosporidium to host cells, the movement and gliding of sporozoites on the surface of host cells, and the formation of vesicles (PV). Micronemes are more numerous in sporozoites, and they play an important role in host cell localization and adhesion. The proteins encoded by the microneme gene family include Gp40 / 15, gp900, P30, and MIC1. Among them, Gp900 protein was first reported by Petersen. It is a mucin-like glycoprotein with a molecular weight greater than 900KDa and a transmembrane domain. GP900 protein is usually stored in micronemes. When Cryptosporidium parvum invades the host, it enters the secretory pathway and is secreted from the micronemes to the surface of the sporozoite membrane. Competitive inhibition experiments using monoclonal antibodies to Cryptosporidium parvum sporozoites showed that Gp900 bound to the surface and apical region of the sporozoite mediated the attachment and invasion of spores. Among the five domains of Gp900, antibodies against domain S34 and domain 3 can inhibit invasion in a dose-dependent manner, which is crucial in the process of sporozoite invasion of host cells. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides an epitope polypeptide of the Gp900 protein of Cryptosporidium parvum and its application.

[0004] An epitope polypeptide of Cryptosporidium parvum Gp900 protein, wherein the epitope polypeptide is an epitope polypeptide of Cryptosporidium parvum Gp900-SAN9 protein.

[0005] Furthermore, the amino acid sequence of the epitope polypeptide is shown in SEQ ID NO.1.

[0006] A nucleic acid encoding the epitope polypeptide according to claim 1, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.2.

[0007] A recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus vector containing the nucleic acid.

[0008] The epitope polypeptide and the nucleic acid are used in preparing a vaccine for preventing or treating Cryptosporidiosis parvum.

[0009] The recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus vector are used in preparing a vaccine for preventing or treating Cryptosporidiosis.

[0010] A vaccine for Cryptosporidiosis parvum, wherein the active ingredient of the vaccine for Cryptosporidiosis parvum is at least one of the following substances:

[0011] A polypeptide epitope of the protein of Cryptosporidium parvum Gp900-SAN9, the amino acid sequence of which is shown in SEQ ID NO.1;

[0012] or a nucleic acid encoding the amino acid sequence of SEQ ID NO.1, whose nucleotide sequence is shown in SEQ ID NO.2;

[0013] Or the nucleic acid shown in SEQ ID NO.2;

[0014] Or the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus vector.

[0015] Furthermore, the vaccine is a long polypeptide epitope vaccine.

[0016] The epitope polypeptide is used to prepare CD8 + T lymphocytes and CD4 + Application in T lymphocyte proliferation agent.

[0017] The nucleic acid or the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus vector is used in the preparation of CD4 + Application in T lymphocyte proliferation agent.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] The present invention provides an epitope polypeptide of Cryptosporidium parvum Gp900 protein and its application. The polypeptide has good affinity with H-2Db molecule, indicating that it has corresponding immunological function, and provides a new direction for preparing Cryptosporidium parvum vaccine based on T cell epitope. The polypeptide has good affinity with H-2Db molecule, indicating that it has corresponding immunological function, and provides a new direction for preparing Cryptosporidium parvum vaccine based on T cell epitope. The polypeptide has good affinity with H-2Db molecule, indicating that it has good ... + T cells and CD4+ T cell immune response showed excellent stimulatory response, which induced CD8 + T and CD4 + Compared with various negative controls, the IFN-γ produced by T cells was significantly different, P < 0.01; the polypeptide can also stimulate the production of polypeptide-specific cytokine TNF-α in the spleen cells of C57BL / 6N mice, and compared with various negative controls, P < 0.01, which is significantly different. This shows that the T cell epitope polypeptide Gp900-SAN9 of Cryptosporidium parvum can effectively stimulate the body's response as a vaccine active substance, and specifically secrete IFN-γ and TNF-α, and has great development and application potential in the field of Cryptosporidium parvum specific immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0021] Figure 1 This is the SDS-PAGE identification diagram of the small amount of induced expression of H-2Db and mβ2m proteins in Example 2 of the present invention;

[0022] Figure 2 The molecular sieve chromatography results and electrophoresis identification after renaturation of H-2Db, mβ2m and epitope polypeptide Gp900-SAN9 in Example 2 of the present invention; wherein A is the molecular sieve chromatography result after renaturation of H-2Db, mβ2m and epitope polypeptide Gp900-SAN9; B is the ion exchange chromatography result after renaturation of H-2Db, mβ2m and Gp900-SAN9 polypeptide; the illustrations in Figures A and B are the SDS-PAGE identification results after molecular sieve chromatography;

[0023] Figure 3 The ELISpot technique of Example 3 of the present invention is used to detect peptide-specific T cells in C57BL / 6N mouse spleen cells; wherein A is mouse spleen CD8 + T cells (1×10 6 ) After culturing for 24 hours with peptide stimulation and without peptide control, antigen-specific cells secreting IFN-γ formed spots; B is the peptide-specific CD8 + T cell ratio analysis, the vertical axis is the number of spot-forming cells, with spot-forming cells (SFC / 10 6 CD8 + IFN-γ +T cells showed extremely significant differences compared with the negative control group, and there was also an extremely significant difference between in vitro addition of peptide stimulation and no addition of peptide (***p<0.001);

[0024] Figure 4 Flow cytometry and intracellular cytokine staining techniques are used to detect polypeptide-specific IFN-γ in Example 3 of the present invention. + T cell results; A is the CD8 T cell expression in spleen cells of mice immunized with Gp900-SAN9 peptide with or without 10 mg / mL Gp900-SAN9 peptide. + IFN-γ + Flow cytometry of T cell ratio; B is Gp900-SAN9 peptide-specific CD8 + IFN-γ + T is the statistical analysis; C is the CD4 + IFN-γ + Flow cytometry of T cell ratio; D is Gp900-SAN9 peptide-specific CD4 + IFN-γ + Statistical analysis of T;

[0025] Figure 5 Flow cytometry and intracellular cytokine staining techniques are used to detect polypeptide-specific TNF-α in Example 3 of the present invention. + T cell results; A is the CD4 T cell count of spleen cells from mice immunized with Gp900-SAN9 peptide with or without 10 mg / mL Gp900-SAN9 peptide + TNF-α + Flow cytometry of T cell ratio; B is Gp900-SAN9 peptide-specific CD4 + TNF-α + T is the statistical analysis; C is the Gp900-SAN9 peptide-specific CD8 + TNF-α + Statistical analysis of T. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0027] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0028] Sources:

[0029] C57BL / 6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0030] Cryptosporidium parvum and Cryptosporidium tayze: donated by Professor Zhang Longxian's team from Henan Agricultural University.

[0031] Amino acid sequence of Gp900-SAN9 epitope polypeptide (SEQ ID NO.1)

[0032] SANTNFLLV

[0033] Nucleotide sequence of Gp900-SAN9 epitope polypeptide (SEQ ID NO.2)

[0034] agtgcaaacaccaacttcttgttggta

[0035] Amino acid sequence of Gp900 (SEQ ID NO.3)

[0036] MVNIKVSSSA IALVAVIMNP LFSLAFKSSN RLEMRIESSG AVSNEKFVIPSLPSDLDPTTFLLIDSTGKK FSPYTGKHAD ASTTSSAYSA PFELDVSGVPIEPNTRRMVD PVSLMLFDNS TGVMYDPNTNSILEGSIAGI RSESCIVSEL NFTSTTGFTTDTSMNWPVSI TSGELKDPNK QATISGSRSC GWKQGYSIDSSTGFRVDSITGLPTDPYPNC PFNPVTGNLV SRSTGKTIPN TYAGVYRSNE TKTTEPSANTNFLLVDPKINAPCNSENSFE QVQIFDMGSK VYIPYTKCVG VKHTTTTTTTTTTTTTTTTT TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTT TTTTTTTTTTKKPTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTTTTTTTTKKPT TTTTTTTTTTTTTTTTTTTT TTTTTTTTKK PTTTTTTTTT TTTTTTTTTT TTTTTTTTTTTTTTATTTTKKPTTTTTTTT TTTTKKPTTT TTATTTTTTS ETESVIKPDE WCWLEKNGECEAKGATYVGVIGKDGRIENG MAFTMIPNDD THVRFRFKVK DVGNTISVRCGKGAGKLEFP DRSLDFTIPP VAGHNSCSIIVGVSGGGKIH VSPYGSKDVS LISAPIQPCELFNEVYCDTC TAKYGAIHSG YQTSADFVTT TTAKPTTTTTGAPGQPTTTTTGSPSKPTTT TTTKATTTTT ILNPIITTTT QKPTTTTTTK VPGKPPIATTTTTLKPIVTTTTTKATTTTT TTVPTTTTTT KRDEMTTTTT PLPDIGDIEI TPIPIEKMLDKYTRMIYDYNSGLLLDSNDE PIPGSQAGQI ADTSNLFPVQ THKSTGLPID PMVGLPFDPKSGNLVHPYTNQTMSGLSVSY LAAKNLTVDT DETYGLPIDT LTGYPLDPVSLIPFNPETGELFDPISDEIM NGTIAGIVSGISASESLLSQ KSAPIDPATN MVVGEFGGLLNPATGVMIPG SLGPSEQTPF SPEIEDGGII PPEVAAANADKFKLSIPPSV PESIPEKDQKIDSISELMYD IESGRLIGQV SKRPIPGSIA GDLNPIMKTP TQTDSVTGKPIDPTTGLPFNPPTGHLINPT NNNTMDSSFA GAYKYAVSNG IKTDNVYGLP VDEITGLPKDPVSDIPFNSTTGELVDPSTG KPINNYTAGI VSGKRGLPPI EDENGNLFDP STKLPIDGNNQLVNPETNST VSGSTSGSTKPKPGIPVNGG GVVPDEEAKD QADKGKDGLIVPPTNSINKD PVTNTQYSNT TGNIINPETG KVIPGSLPGSLNYPSFNTPQ QTDEITGKPVDTVTGLPYDP STGEIIDPAT KLPIPGSVAG DEILTEVLNI TTDEVTGLPIDLETGLPRDPVSGLPQLPNG TLVDPSNKKP IPGSHSGFIN GTSGEQSHEK DPSTGKPLDPNTGLPFDEDSGSLINPETGD KLQGSHSGTF MPVPGKPQGE NGGIMTPEQILEALNKLPTS NEVNISPRPS SDAVPDRPTNTWWNKISGQT FQVDGKKTIPGSAASVIHTA LGTPTQTDPT TGLPSDPSTG LPFIPGFNVL VDPQTGEQIKGSVPYVSLYVKEKNIVTEAA YGLPVDPKTG FPIDPISYLP FAKNGELIDP ISGKYFSGSIAGFISGKAGSQSKSSDESGN PIDPSTNMPY DPKTGKLIDP ESGIAIDNSV SGVFATVPGTAAPKKGGVIPESVAAEAAKK YFAANVEGEG EGEEVPPPPE SSSNIAIQAA GGASAAVGLVAAVGAWYASRNRQEGEDDDD YQMDLKQNMK KKRKKRVMKQ QMKLLLQLSVIHHSGTNLKR RKDFSNSKKF RI

[0037] Nucleotide sequence of Gp900 (SEQ ID NO.4)

[0038]

[0039]

[0040]

[0041]

[0042] Example 1 Selection and Synthesis of Cryptosporidium parvum Protein Epitope Peptides

[0043] The main pathogenic proteins and surface proteins of Cryptosporidium parvum Gp40 / 15, CSL, Gp900 and Cp23 were scanned and predicted by the following website (https: / / services.healthtech.dtu.dk / service.php?NetMHC-4.0). A total of 38 epitope peptides were obtained, and the results are shown in Table 1. The predicted peptide affinity is expressed as IC50 (nM), and the peptide affinity range is between 6.88-1423.8nM. Another parameter (%Rank) value indicates the rank of the peptide predicted to be bound by MHC. The smaller the %Rank value, the stronger the ability of the peptide to be bound by the corresponding MHC molecule. The %Rank values ​​of the 38 peptides are all less than 0.5. Since the experimental data are too numerous, only the Gp900 epitope polypeptide (Gp900-SANTNFLLV, denoted as Gp900-SAN9) related to the present invention is described here. The %Rank value between the Gp900-SAN9 polypeptide and MHC is 0.01, indicating that it has a strong affinity with the MHC molecule. The polypeptide was synthesized by solid phase peptide synthesis (Fmoc / tBu strategy) and purified by HPLC after synthesis, and its purity was 90%.

[0044] Table 1 Prediction of Cryptosporidium parvum proteins

[0045]

[0046]

[0047] The amino acid sequence of the Gp900-SAN9 epitope polypeptide is shown in SEQ ID NO.1.

[0048] The nucleotide sequence of the Gp900-SAN9 epitope polypeptide is shown in SEQ ID NO.2.

[0049] The amino acid sequence of Cryptosporidium parvum Gp900 protein is shown in SEQ ID NO.3.

[0050] The nucleotide sequence encoding the Cryptosporidium parvum Gp900 protein is shown in SEQ ID NO.4.

[0051] Example 2 Protein in vitro renaturation experiment to detect the affinity of epitope peptide Gp900-SAN9 with mouse H-2Db molecule and light chain β2m (mouseβ2m, mβ2m) molecule

[0052] 1. Small-scale induced expression of recombinant expression plasmid

[0053] (1) The recombinant plasmids of the constructed H-2Db and mβ2m genes were transformed into E. coli BL21 (DE3), and the positive colonies were selected and cultured at 37°C and 200 r / min for 7-10 h, and then stored at 4°C as the mother bacterial liquid;

[0054] (2) The mother bacterial liquid was inoculated into 3 mL of Amp / LB medium at a ratio of 1%-5%, and cultured in a shaking incubator at 200 r / min at 37°C until the OD600 reached 0.4-0.8;

[0055] (3) Pipette 500uL of culture medium into a 1.5mL centrifuge tube as an uninduced control, add 2.5μL of 1MIPTG to the remaining sample (to a final concentration of 1mM), and continue culturing for 6h at 37°C and 160-170r / min;

[0056] (4) Pipette 500uL of culture medium into a 1.5mL vial, centrifuge it together with the uninduced control sample (12000r / min, 3min), and discard the supernatant; resuspend it with 30μL 1×PBS, add 10uL 4×SDS loading buffer, mix well, and boil for 5-10min. Centrifuge at 12 000r / min for 1min. Pipette the supernatant for SDS-PAGE analysis. The SDS-PAGE identification of the small amount of induced expression of H-2Db protein is shown in the figure below. Figure 1 As shown in A, the SDS-PAGE identification of mβ2m protein induced expression in small amounts is shown in Figure 1 As shown in B, it can be seen from the figure that the H-2Db molecule is induced to express in a small amount with a molecular weight of about 32KDa, and the mβ2m has a molecular weight of about 12KDa.

[0057] 2. Folding and renaturation of H-2Db, mβ2m and Gp900-SAN9 peptides

[0058] The inclusion body proteins of mouse MHC class I heavy chain (H-2Db) and light chain (mβ2m) and Gp900-SAN9 polypeptide were added to the inclusion body refolding reagent (Refolding Buffer) at a molar ratio of 1:1:3. The specific process is as follows:

[0059] (1) Precool the inclusion body refolding reagent (usually 500 ml system, which can be determined according to the refolding effect) in a 4°C refrigerator (or cold storage). The refolding process is carried out at 4°C (4°C refrigerator or cold storage);

[0060] (2) Place the beaker containing the inclusion body refolding reagent on a magnetic stirrer, add a magnetic rotor, and set an appropriate stirring speed (preferably 100-200 rpm). Take a 5 mL syringe and fix it on the beaker;

[0061] (3) Take 1 mL of mβ2m inclusion bodies (30 mg / ml) dissolved in guanidine hydrochloride and add it to a syringe, slowly drip it into the inclusion body refolding reagent, and stir slowly for 6-8 hours;

[0062] (4) 5 mg of Gp900-SAN9 peptide was dissolved in 200 μL DMSO and then added directly to the inclusion body refolding reagent using a pipette;

[0063] (5) After slowly stirring for 30 minutes, add 3 mL of H-2Db inclusion bodies (30 mg / ml) into the syringe and slowly stir for 12 hours. This can appropriately extend the action time.

[0064] 3. Concentration of complex proteins

[0065] After the renaturation is completed, transfer the renatured solution to a pressure stirring concentration cup in a 4°C refrigerator and concentrate it using a 10KDa ultrafiltration membrane using the nitrogen pressure method (0.4Kpa). When the solution is concentrated to a volume of about 30mL, add 100mL of pre-cooled molecular sieve buffer. When the solution volume is concentrated to about 50mL again, centrifuge at 4°C, 12,000r / min for 10min. Transfer the supernatant to a 10KD ultrafiltration concentration tube and continue to concentrate it at 4°C, 2400r / min. Take it out when the concentrated volume is about 5mL.

[0066] 4. Identification of complex proteins by molecular sieve chromatography and ion exchange chromatography

[0067] The Superdex 200 16 / 60HiLoad gel chromatography column was balanced with molecular sieve buffer to the baseline (generally 1 ml / min, about 2 hours). The concentrated and liquid-changing complex protein sample was centrifuged to remove the precipitate (4°C, 12000 rpm, 10 min). The loading pump of the fast protein liquid chromatography (FPLC) was washed with molecular sieve buffer, and the supernatant was injected. The column was run at an appropriate flow rate (generally 1 ml / min), and the renaturation effect was detected according to the results of molecular sieve chromatography. The target peak protein of the corresponding molecular weight was collected and identified by SDS-PAGE. The protein peak collection identified as the target protein (complex) was further analyzed with an anion exchange column Resource Q. First, the column was equilibrated with ion exchange solution B (10mM Tris-HCl, 1MNaCl, pH 8.0), and then the column was equilibrated with ion exchange solution A (10mM Tris-HCl, 10mM NaCl, pH 8.0) and the sample was loaded. After the sample was loaded on the column, a linear gradient elution was performed for 60 minutes using an eluent composed of ion exchange solution A and ion exchange solution B. Within the 60 minutes, the volume ratio of ion exchange solution A in the eluent linearly decreased from 100% to 50%, and the volume ratio of ion exchange solution B in the eluent linearly increased from 50% to 100%. The elution peaks were collected and identified by SDS-PAGE. The complex of H-2Db protein, mouse mβ2m protein and Gp900-SAN9 was recorded as H-2Db / Gp900-SAN9. The results are shown in Figure 2. Figure 2 As shown, A is the molecular weight of H-2Db, mβ2m and Gp900-SAN9, which is about 32KDa, mβ2m is about 12KDa, and Gp900-SAN9 is just a short peptide, which can be ignored.) The molecular sieve chromatography results after renaturation are as follows. Figure 2 A shows that there are three main peaks in protein chromatography under the condition of ultraviolet absorption at 280nm, among which peak 1 is the multimer peak of H-2Db heavy chain, and peak 2 (target protein peak) is the trimolecular complex peak formed by H-2Db, mβ2m and polypeptide. The proteins at the peaks are collected and analyzed by 12% SDS-PAGE electrophoresis. Figure 2 Panel A shows the molecular sieve chromatogram after renaturation of Gp900-SAN9 polypeptide and the SDS-PAGE identification results of the collected protein peaks. The results show that peak 2 at 82 mL elution volume is the H-2Db / Gp900-SAN9 / mβ2m complex monomer, and its protein molecular weight is about 44 KDa, indicating that the polypeptide has a certain in vitro affinity with H-2Db; peak 3 is around 105 mL elution volume, which is the mβ2m monomer protein.

[0068] Peak 2 was collected and the H-2Db / Gp900-SAN9 / mβ2m complex monomer was further identified by ion exchange chromatography. The identification results are shown in Figure 2B, The peptide complex forms a higher target peak at a conductivity of 12-16 mS / cmx, indicating that the peptide has a higher stability in binding to the H-Db molecule in vitro.

[0069] Example 3 Detection of specific CTL immune response function in experimental animals

[0070] 1. Immunization of experimental animals

[0071] (1) Dissolve 5 mg of the Gp900-SAN9 epitope polypeptide prepared in Example 1 with 500 μL of DSMO to a final concentration of 10 mg / mL. Take 50 μL of the dissolved polypeptide and dilute it 10 times with PBS buffer to 5500 μL, add an equal amount of immune adjuvant, and emulsify it with a handheld high-speed emulsifier until the liquid is cheese-like and there is no obvious liquid flow when the centrifuge tube is inverted.

[0072] (2) Female C57BL / 6 mice aged 6-8 weeks were selected and divided into a polypeptide immunization group, an oocyst gavage group, a Freund's adjuvant control group, and In the control group, 6-10 mice were selected in each group, and the oral gavage group was not treated with peptide immunization. Each mouse in the peptide immunization group and the oral gavage group was gavaged with 2×10 5 The peptide immunization group was immunized with peptide by subcutaneous injection at the back of the neck. Each mouse was immunized with 50 μg of peptide. Each immunization was separated by 14 days. The first immunization used Freund's complete immune adjuvant, and the second and third immunizations used Freund's incomplete adjuvant. Seven days after the last immunization, 5 mice were taken from each group to isolate the spleen CD8 + T cells were subjected to ELISpot assay and intracellular cytokine staining assay.

[0073] 2. Isolation of Mouse Spleen Cells

[0074] The mice were killed by cervical dislocation and placed in 75% alcohol for later use; the spleen was separated and peeled off slowly with forceps; the separated spleen was added to 1640 culture medium and ground with a syringe tip using a 70 μm cell sieve; the grinding liquid was slowly aspirated into a centrifuge tube, and centrifuged at 1500 rpm (350 g) at 4°C for 5 min. After centrifugation, the supernatant was discarded and the remaining liquid was flicked 5 times with the index finger to pick up the cells; 10 ml of cell lysis solution was added, and the cells were lysed at room temperature for 10 min and centrifuged at 1500 rpm for 5 min; the cells were washed twice with 1640 culture medium to remove red blood cells and connective tissue; after centrifugation, the supernatant was carefully transferred to a new 15 ml centrifuge tube, and 10 ul of cells were counted (bright round white spots observed under a microscope).

[0075] 3. Magnetic Bead Method for Isolation of Mouse Splenic CD8 + T cells

[0076] According to MojoSort TM Mouse CD8 + According to the instructions of the T Cell Isolation Kit, the concentration of spleen cells was adjusted to 1×10 8 / mL, resuspend the cells in an appropriate volume of MojoSort TM Buffer, centrifuge at 1000r / min for 5min. Take 100μL cell suspension (about 10 7 cells) were placed in new flow cytometry tubes, 10 μL of biotin-antibody cocktail was added to each tube, mixed thoroughly, and incubated on ice for 15 min; 10 μL of streptavidin nanobeads was added to each tube, vortexed and mixed, and incubated on ice for 15 min; 2.5 mL of MojoSort TM Buffer, place the flow tube in the center of the magnet and let it stand for 5 minutes, then pour out the contents of the tube to obtain the separated CD8 + T cells, using isolated CD8 + T cells were subjected to ELISpot assay.

[0077] 4. Detection of mouse IFN-γ using ELISpot (Enzyme-linked Immunospot Assay)

[0078] Take the prepared lymphocytes (about 10 6 cells) and plated them on the treated ELISpot plates. The reported positive epitope Gp40 / 15-SVF9 of Cryptosporidium parvum was used as the positive peptide control. The adjuvant immunization group and the oral gavage control group were set up respectively. The peptide immunization group was stimulated with the corresponding peptide (at a concentration of 10ug / ml). The positive peptide Gp40 / 15-SVF9 group was stimulated with Gp40 / 15-SVF9 in vitro. The Gp900 peptide group, the adjuvant immunization group and the oral gavage group were all stimulated with Gp900-SAN9 peptide. The CD8 + T cells were cultured in 37℃ CO2 medium for 12-48h. The ELISpot plate could not be moved during the culture period. After the culture, the cells were discarded and washed 5 times with PBS. 200ul was added to each well. Biotinylated antibody (1ug / ml) was added and incubated at 37℃ for 2h. PBS was washed 5 times. HRP-Streptavidin was added and incubated at 37℃ for 1h. PBS was washed 5 times. Substrate was added for color development. After drying at room temperature, the plate was read on an ELISpot reader. A is mouse spleen CD8 + T cells (1×10 6) After 24 h of culture with or without polypeptide stimulation, antigen-specific cells secreting IFN-γ formed spots; Figure 3 B is the difference between the peptide-specific CD8 + T cell ratio analysis, the vertical axis is CD8 under different treatment conditions + The number of IFN-γ spot-forming cells in T cells was expressed as spot-forming cells (SFC / 10). 6 It can be seen from the figure that the CD8 + IFN-γ + T cells showed a significant difference compared with the negative control group, and there was also a significant difference between in vitro peptide stimulation and no peptide stimulation (***p<0.001). There was no significant difference between the proportion of spot-forming cells in the Gp900-SAN9 peptide immunization group and the published Cryptosporidium parvum immunodominant epitope Gp40 / 15-SVF9.

[0079] 5. Intracellular cytokine staining to detect IFN-γ and TNF-α in mice

[0080] (1) Collect and process cells

[0081] Sample wells per 1mL (10 7 The corresponding polypeptide was added to the cells (cells / mL) to a final concentration of 10 μg / mL, and 2uL of cell stimulator (Cell Activation Cocktail) was added to the positive control; the cells were cultured in a 5% CO2 incubator at 37°C for 2 hours, and the blocking agent BFA was added to continue culturing for 6 hours; no polypeptide was added to the negative control, but the blocking agent BFA was added at the same time.

[0082] (2) Cell death and viability staining

[0083] The cell suspension was collected by centrifugation at 1500 r / min for 5 min, and the cells were washed with HBSS; the cells were resuspended in 1 mL HBSS and 1 μL FVS 510 dye was added, and the cells were stained at room temperature in the dark for 15 min. After centrifugation at 1500 r / min for 5 min, the supernatant was discarded and the cells were washed twice by FACS.

[0084] (3) Surface molecular staining

[0085] Take 100 μL FACS, add 1 μL FITC-labeled anti-mouse CD8 monoclonal antibody, 1 μL APC-labeled anti-mouse CD3 monoclonal antibody, 1 μL APC-Cy7-labeled anti-mouse CD4 monoclonal antibody, stain at 4°C in the dark for 30 minutes, add 1900 μL PBS, centrifuge at 1500 r / min for 5 minutes, add 2 mL FACS to resuspend the cells, and wash the cells twice.

[0086] (4) Fixation and membrane disruption

[0087] Add 250 μL of fixative precooled at 4°C, fix the cells for 20 min at room temperature in the dark; add permeabilization wash buffer, 1 mL / tube, react at room temperature in the dark for 20 min; centrifuge at 1500 r / min, 4°C for 5 min, and discard the supernatant.

[0088] (5) Intracellular cytokine staining

[0089] Add 100 μL of membrane permeabilization agent to resuspend the cells, add 1 μL of antibodies PE-IFN-γ and PerCP-TNF-α respectively, and incubate for 30 min; add membrane permeabilization solution to 1 mL, centrifuge at 1500 r / min for 5 min, and discard the supernatant; add 1 mL / tube of FACS washing solution to wash the cells once, centrifuge at 1000 r / min for 5 min, and discard the supernatant; resuspend the cells with 500 μL FACS buffer for flow cytometry detection.

[0090] (6) The gating schemes for flow cytometry are lymphocytes, monocytes, live CD3 + T cells, CD4 + T cells and CD8 + T cells, double-positive T cells that secrete IFN-γ and TNF-α cytokines in CD4 and CD8 cells.

[0091] The results showed that the Gp900-SAN9 polypeptide prepared by the present invention can induce stronger CD8 + T cell immunity was significantly different from that of the negative control group (PBS + Freund's adjuvant) (P<0.001). In order to verify the peptide-specific T cell immune response, 10ug / mL of peptide or no peptide was added to the isolated spleen cells for paired experimental analysis. The experimental results are shown in Figure 4 As shown, Figure 4 A is the CD8 + IFN-γ + Flow cytometry of T cell ratio, Figure 4B is Gp900-SAN9 peptide-specific CD8 + IFN-γ + The results of statistical analysis of T showed that the spleen cells of mice in the Gp900-SAN9 polypeptide immunization group prepared by the present invention could produce memory responses to the in vitro polypeptides, and compared with the group without polypeptide, IFN-γ + CD8 + The proportion of specific T cells increased significantly (P<0.05). + T cell epitope Gp900-SAN9 can also induce IFN-γ production in mice + CD4 + Double positive T cells. Figure 4 C is the CD4 + IFN-γ + Flow cytometry of T cell ratio, Figure 4 D is Gp900-SAN9 peptide-specific CD4 + IFN-γ + Statistical analysis results of T. Figure 4 D shows that in the paired experiment for peptide-specific T cell analysis, the spleen cells of mice in the Gp900-SAN9 peptide immunization group were able to produce memory responses to the in vitro peptides, and compared with those without peptides, IFN-γ + CD4 + There was a very significant difference in the proportion of double-positive T cells (P<0.01).

[0092] TNF-α is a cytokine that can mediate viral, intracellular bacterial and parasitic infections. In addition, T cells that co-produce IFN-γ and TNF can enhance the killing effect on Cryptosporidium parvum. Therefore, based on the analysis of IFN-γ, the CD8 + TNF-α + and CD4 + TNF-α + The data of the proportion of double-positive T cells were analyzed. Figure 5 A. Figure 5 As shown in B, Figure 5 A is the CD4 + TNF-α + Flow cytometry of T cell ratio, Figure 5 B is Gp900-SAN9 peptide-specific CD4 + TNF-α+ The statistical analysis of T showed that Gp900-SAN9 peptide can also induce strong TNF-α + CD4 + The T cell response was significantly different from that in the group without peptide addition (P<0.01). Figure 5 C is Gp900-SAN9 peptide-specific CD8 + TNF-α + Statistical analysis of T. As can be seen from the figure, Gp900-SAN9 induced CD8 + TNF-α + The T cell response was also peptide-specific. The addition of Gp900-SAN9 peptide in vitro was significantly different from the negative control without peptide (P<0.01).

[0093] The T cell epitope polypeptide Gp900-SAN9 of Cryptosporidium parvum of the present invention can be used as an active ingredient, which can not only stimulate mice to produce CD8 + T cells, and produced CD4 + The specific immune response mainly based on T cells further has a killing effect on Cryptosporidium parvum. It is combined with a pharmaceutically acceptable carrier to form a composition, or combined with other pharmacologically active extracts and / or synthetic drugs to form a composition, and then prepared into a Cryptosporidium parvum preventive or therapeutic polypeptide vaccine based on the Gp900-SAN9 epitope according to conventional methods in the pharmaceutical field, which has great development and application potential in the field of Cryptosporidium parvum specific immunotherapy.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. An epitope polypeptide of Cryptosporidium parvum Gp900 protein, characterized in that: The epitope polypeptide is an epitope polypeptide of Cryptosporidium parvum Gp900-SAN9 protein.

2. The epitope polypeptide according to claim 1, characterized in that The amino acid sequence of the epitope polypeptide is shown in SEQ ID NO.

1.

3. A nucleic acid encoding the epitope polypeptide according to claim 1, characterized in that The nucleotide sequence of the nucleic acid is shown as SEQ ID NO.

2.

4. A recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant viral vector containing the nucleic acid according to claim 3.

5. Use of the epitope polypeptide according to claim 1 and the nucleic acid according to claim 3 in the preparation of a vaccine for preventing or treating Cryptosporidiosis parvum.

6. Use of the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant viral vector according to claim 4 in the preparation of a vaccine for preventing or treating Cryptosporidiosis.

7. A vaccine for Cryptosporidiosis parvum, characterized in that: The active ingredient of the vaccine for Cryptosporidiosis parvum is at least one of the following substances: A polypeptide epitope of the protein of Cryptosporidium parvum Gp900-SAN9, the amino acid sequence of which is shown in SEQ ID NO.1; or a nucleic acid encoding the amino acid sequence of SEQ ID NO.1, whose nucleotide sequence is shown in SEQ ID NO.2; Or the nucleic acid shown in SEQ ID NO.2; Or the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant viral vector according to claim 4.

8. The vaccine according to claim 7, characterized in that The vaccine is a long polypeptide epitope vaccine.

9. The epitope polypeptide according to claim 1 is used in the preparation of CD8 + T lymphocytes and CD4 + Application in T lymphocyte proliferation agent.

10. The nucleic acid of claim 3 or the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant viral vector of claim 4 in the preparation of CD4 + Application in T lymphocyte proliferation agent.

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