阅读说明：本技术 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 (Cryptosporidium parvum Gp40/15 protein epitope polypeptide and adenovirus vector vaccine thereof ) 是由 王永立 樊淑华 李兵 高明慧 赵孟春 李佳玥 杨同文 于 2021-10-09 设计创作，主要内容包括：本发明涉及一种微小隐孢子虫Gp40/15蛋白的免疫优势T细胞表位多肽,该表位多肽的氨基酸序列如SEQ ID NO.3所示。该多肽与H-2Kb具有较高的体外亲和力且具有一定的稳定性。涉及一种重组腺病毒载体疫苗,该疫苗表达微小隐孢子虫表面糖蛋白gp40/15核苷酸,病毒载体疫苗经小鼠免疫后,能显著刺激小鼠的T细胞免疫应答。其所诱导CD8+T cell产生的IFN-gamma,与各种阴性对照比较,P＜0.01,具有显著差异。多肽免疫组特异性双阳性T细胞比例明显高于阴性对照,说明gp40/15蛋白及其相关多肽具有相应的生物免疫学功能,成为新型的微小隐孢子虫疾病表位疫苗或腺病毒载体疫苗。(The invention relates to an immunodominant T cell epitope polypeptide of cryptosporidium parvum Gp40/15 protein, and the amino acid sequence of the epitope polypeptide is shown as SEQ ID NO. 3. The polypeptide has high in vitro affinity with H-2Kb and certain stability. Relates to a recombinant adenovirus vector vaccine, which expresses cryptosporidium parvum surface glycoprotein gp40/15 nucleotide, and the virus vector vaccine can remarkably stimulate the T cell immune response of mice after the mice are immunized. Compared with various negative controls, the IFN-gamma generated by the induced CD8+ T cell has the P of less than 0.01 and has obvious difference. The specific double-positive T cell ratio of the polypeptide immunity group is obviously higher than that of a negative control, which shows that the gp40/15 protein and related polypeptides thereof have corresponding biological immunological functions and become a novel cryptosporidium parvum disease epitope vaccine or an adenovirus vector vaccine.)

1. The cryptosporidium parvum Gp40/15 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 3.

2. A nucleic acid encoding the epitope polypeptide of Cryptosporidium parvum Gp40/15 protein according to claim 1.

3. The nucleic acid of claim 2, wherein the nucleotide sequence of the nucleic acid is set forth in SEQ ID No. 4.

4. A recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector comprising the nucleic acid of claim 2 or 3.

5. Use of the cryptosporidium parvum Gp40/15 protein epitope polypeptide of claim 1, the nucleic acid of claim 2 or 3 for the preparation of a vaccine for the prevention or treatment of cryptosporidium parvum disease.

6. Use of the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector of claim 4 for the preparation of a vaccine for the prevention or treatment of cryptosporidiosis.

7. The cryptosporidium parvum vaccine is characterized in that the active component of the cryptosporidium parvum vaccine is at least one of the following substances:

a. the cryptosporidium parvum Gp40/15 protein epitope polypeptide has an amino acid sequence shown in SEQ ID No. 3;

b. nucleic acid encoding epitope polypeptide with amino acid sequence shown in SEQ ID NO. 3;

c. nucleic acid with the nucleotide sequence shown as SEQ ID NO. 4;

d. the recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium, or recombinant viral vector of claim 4.

8. The Cryptosporidium parvum vaccine of claim 7, wherein the vaccine is an adenovirus vaccine.

9. The use of the cryptosporidium parvum Gp40/15 protein epitope polypeptide of claim 1 in the preparation of CD8+ T lymphocyte proliferation agent.

10. Use of the nucleic acid of claim 2 or 3, the recombinant expression vector, the expression cassette, the transgenic cell line, the recombinant bacterium or the recombinant viral vector of claim 4 for the preparation of a CD8+ T lymphocyte proliferation agent.

Technical Field

The invention belongs to the technical field of biological molecules, and relates to an adenovirus vector vaccine for coding cryptosporidium parvum Gp40/15 protein and a T cell epitope polypeptide thereof.

Background

Cryptosporidium parvum belongs to the Class of sporozoea (Class Sporozoa) of the phylum apicomplexa, first reported and named by Tyzzer in 1912, and is an important zoonotic protozoan. Among the 31 reported classes of zoonotic cryptosporidium, cryptosporidium parvum is responsible for infection in humans and most mammals, and the host range for cryptosporidium parvum is the most widespread, including humans, bovines, various rodent mammals, camelids, equines, canines, non-human primates, and marine mammals and fish. The cryptosporidium parvum sporozoite surface adhesion protein Gp60 is a main antigen on the surface of cryptosporidium parvum sporozoites, which is simultaneously discovered and reported by three research teams of Cevallos et al and Strong et al in 2000, and is an effective target antigen for specifically preventing and treating cryptosporidium parvum infection. The cryptosporidium parvum precursor glycoprotein Gp60 is proteolytically processed to yield two mature glycoproteins Gp40 and Gp15, the major proteins involved in the attachment and invasion of host cells by cryptosporidium parvum sporozoites. Gp15, the carboxy-terminal conserved region of Gp60, is anchored to the sporozoite surface by Glycosylphosphatidylinositol (GPI), while Gp40, the amino-terminal polytropic region, has the sequence characteristic of an α -N acetylgalactosamine residue, binds to the human small intestinal epithelial cell receptor. Research shows that monoclonal antibody recognizing Gp40 has the capacity of preventing cryptosporidium parvum from invading host cell; the recombinant Gp40/15 protein can stimulate mice to generate immunoprotection.

Cryptosporidium parvum is an intracellular parasitic protozoan, and its CTL epitope is presented by MHC i molecules to CD8+ T cells, thereby activating the cellular immune response of the body. Cytotoxic T Lymphocytes (CTL) -mediated specific cellular immune responses play a key role in the body's process against intracellular microbial infections. After the cryptosporidium parvum oocysts infect cells, proteins such as Gp40/15, Cp15 and Cp23 on the surfaces of the cryptosporidium parvum oocysts are degraded into polypeptides with 8-11 amino acids through a series of enzymolysis processes in the infected cells, and then the polypeptides are combined with corresponding Major Histocompatibility Complex (MHC) class I molecule antigen polypeptide binding grooves to form MHCI-antigen peptide complexes and are presented on the surfaces of the infected cells. The mhc i-antigen peptide complex (pMHCI) is recognized by and binds to a receptor (TCR) expressed on the surface of T cells. Cytotoxic T Lymphocytes (CTL) can lyse infected cells in the gastrointestinal tract by releasing perforin or granzyme, release immature cysts, and cut off the proliferation of cryptosporidium in cells, thereby effectively controlling the infection of cryptosporidium. Studies have shown that cellular immune responses play a more important role in resisting cryptosporidium infection than humoral immune responses, and that both CD4+ and CD8+ T cells play an important role in resisting cryptosporidium parvum (c.parvum) immunity. As found in functional studies by Pantenburg et al on the clearance of Cryptosporidium parvum infection in intestinal epithelial cells by human CD8+ T cells, human HLA I molecules activate the CD8+ T cell immune response by presenting an immunodominant epitope of the cryptosporidium Gp15 protein.

Disclosure of Invention

In view of the above, the present invention aims to provide an adenovirus vector vaccine encoding cryptosporidium parvum Gp40/15 protein, and related epitope polypeptide thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a cryptosporidium parvum Gp40/15 protein epitope polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 3.

2. Nucleic acid of cryptosporidium parvum Gp40/15 protein epitope polypeptide with the amino acid sequence shown in SEQ ID NO. 3.

Further, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 4.

3. Contains a nucleic acid of cryptosporidium parvum Gp40/15 protein epitope polypeptide with the coding amino acid sequence shown as SEQ ID NO.3, an expression vector, a transgenic cell line, a recombinant bacterium or a recombinant virus vector.

Further, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 4.

The nucleic acids of the invention can be in the form of RNA (e.g., mRNA, hnRNA, tRNA or any other form) or in the form of DNA (including, but not limited to, cDNA and genomic DNA produced by cloning or synthetically, or any combination thereof). The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also referred to as the sense strand, or may be the non-coding strand, also referred to as the antisense strand.

4. The application of the cryptosporidium parvum Gp40/15 protein epitope polypeptide with the amino acid sequence shown in SEQ ID NO.4 in the preparation of vaccines for preventing or treating cryptosporidium parvum disease.

Further, the application of the nucleic acid of the cryptosporidium parvum Gp40/15 protein epitope polypeptide with the coded amino acid sequence shown as SEQ ID NO.3 in the preparation of vaccines for preventing or treating cryptosporidium parvum disease.

5. The recombinant expression vector, the expression cassette, the transgenic cell line, the recombinant bacterium or the recombinant virus vector in the technical scheme 3 is applied to the preparation of the vaccine for preventing or treating the cryptosporidiosis.

6. A cryptosporidium parvum vaccine, the active ingredient of the cryptosporidium parvum vaccine is at least one of the following substances:

a. the amino acid sequence of the polypeptide of the cryptosporidium parvum Gp40/15 protein epitope is shown in SEQ ID NO. 3;

b. nucleic acid encoding epitope polypeptide with amino acid sequence shown in SEQ ID NO. 3;

c. nucleic acid with the nucleotide sequence shown as SEQ ID NO. 4;

d. the recombinant expression vector, the expression cassette, the transgenic cell line, the recombinant bacterium or the recombinant virus vector in the technical scheme 3.

Further, the vaccine is an adenovirus vaccine.

7. The technical scheme 1 is the application of the cryptosporidium parvum Gp40/15 protein epitope polypeptide in the preparation of CD8+ T lymphocyte proliferation agents.

8. The use of a nucleic acid according to claim 2 or 3, a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant viral vector according to claim 4 for the preparation of a CD8+ T lymphocyte proliferation agent.

The invention has the beneficial effects that: the invention provides cryptosporidium parvum Gp40/15 protein epitope P2, which can cause specific T cell immune response in a mouse body, has certain in vitro affinity and stability with H-2Kb molecules in vitro, shows that the cryptosporidium parvum Gp40/15 protein epitope P2 has a corresponding biological immunological function, and provides a new direction for preparing cryptosporidium parvum vaccines. The Gp40/15 adenovirus vector vaccine is prepared to immunize C57BL/6N mice, and experimental results show that the polypeptide P2 can stimulate the spleen cell specific CTL immune response of the C57BL/6N mice, compared with other polypeptides and negative control, the IFN-gamma generated by induced CD8+ T cells has the P of less than 0.01 and has obvious difference, which indicates that the polypeptide P2 as a vaccine active substance can effectively stimulate the organism reaction and stimulate the specific T cells to specifically secrete the IFN-gamma. Detecting the proportion of FITC marked CD8+ T and PE marked IFN-gamma double positive cells by flow cytometry, wherein the negative control group is 0.087%; the P2 polypeptide stimulated group was 1.23%, with very significant differences. The tetramer protein of the P2 polypeptide is prepared in vitro, and the detection result shows that the proportion of the vaccine immunization group polypeptide specificity double-positive T cells is 0.84 percent and is obviously higher than that of a negative control (PBS immunization group) by 0.23 percent. The adenovirus vector vaccine prepared by the cryptosporidium parvum Gp40/15 protein epitope polypeptide P2 nucleic acid can induce and generate CTL with killing activity to target cells, and has great development and application potential in the field of cryptosporidium parvum specific immunotherapy.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the results of molecular sieve chromatography and ion exchange chromatography after renaturation of H-2Kb, m beta 2m and polypeptide P2. Wherein A is a molecular sieve chromatography result after renaturation of H-2Kb, m beta 2m and polypeptide P2; b is the result of ion exchange chromatography after renaturation of H-2Kb, m beta 2m and polypeptide P2; the insert picture is the result of SDS-PAGE identification after molecular sieve chromatography.

FIG. 2 is a SDS-PAGE electrophoretic identification of biotinylated and tetrameric P2 polypeptide proteins; wherein A is a biotinylation result identification picture; b is an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification picture of tetrameric protein of the polypeptide P2.

FIG. 3 shows the results of the identification of Cryptosporidium epitopes by ELISPOT. Wherein the abscissa is 4 polypeptides and positive control polypeptide (P.berghei, PbTRAP 130-138: SALLNVDNL), the ordinate is spot-forming cell number, and spot-forming cells (SFC/10) are spot-forming cells (spot-forming cells), and the negative control (cells not stimulated by added polypeptide)⁶And (4) showing. The number of spots generated by the polypeptide P2(Gp40/15-AIF9) is higher than that of the positive control polypeptide, and the spots are also significantly different (P < 0.01) compared with the other three polypeptides.

FIG. 4 shows the result of intracellular cytokine staining with P2 polypeptide. Wherein A is a spot produced in the absence of added polypeptide; b is a spot generated by polypeptide stimulation, and C is a cell surface FITC-labeled CD8 antibody of P2 polypeptide and a PE-labeled IFN-gamma double staining result.

FIG. 5 is a tetramer assay to detect P2-specific T cells. Wherein A is blank as staining control; b is FITC labeled CD8+ antibody single staining control; c is a single stain control of PE-labeled tetramer; d is the double staining result of the polypeptide P2 stimulation group; f is the double staining result of the negative control.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 selection and Synthesis of Cryptosporidium parvum protein epitope Polypeptides.

The main pathogenic proteins of cryptosporidium parvum and the surface proteins Gp40/15, CSL, Gp900, Cp15 and Cp23 were scanned and predicted by the following website (https:// services. health. dtu. dk/service. php. NetMHC-4.0). A total of 203 epitopes were obtained, 107H-2 Kb-restricted polypeptide epitopes and 123 HLA-A0201-restricted polypeptide epitopes, with predicted polypeptide affinities expressed as IC50(nM) ranging from 2.9 to 1179.35nM, including 17 HLA-A0201 and H-2Kb crossover epitopes. (strong binding polypeptide SB, affinity threshold% Rank < 0.500), weak binding polypeptide WB, 0.500 < affinity threshold% Rank < 2.000). The polypeptide was synthesized by solid phase peptide synthesis (Fmoc/tBu strategy) and purified by HPLC after synthesis to a purity of 98%. After synthesis, experiments are further adopted to verify the immunogenicity of the compound. The experimental data are too numerous, and only the epitope polypeptide P2(Gp40/15-AIF9) relevant to the invention is described here, the amino acid sequence of the polypeptide is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4. The polypeptide P2 is derived from Gp40/15 protein. Table 1 is a partially predicted epitope.

TABLE 1

The amino acid sequence of the polypeptide P2 is shown as SEQ ID NO.3 in the sequence table, and the nucleotide sequence is shown as SEQ ID NO.4 in the sequence table. The sequences SEQ ID NO.1 and SEQ ID NO.2 are the amino acid sequence and the nucleotide sequence of the cryptosporidium parvum Gp40/15 protein, respectively.

Example 2 protein in vitro renaturation experiment to detect the affinity of P2 epitope polypeptide and mouse light chain beta 2m (mouse beta 2m, m beta 2m) molecule

Folding renaturation of mono, H-2Kb, mbeta 2m and polypeptide P2

Inclusion body proteins of mouse MHC class I heavy chain (H-2Kb) and light chain (m beta.2m) and polypeptide P2 were added to inclusion body renaturation reagent (reforming Buffer) at a molar ratio of 1:1: 3. The specific process is as follows:

(1) the inclusion body renaturation reagent (generally 500ml system, can be according to the conventional dependence on renaturation effect) is placed in a refrigerator (or a cold storage) for precooling at 4 ℃. The renaturation process is carried out at 4 ℃ (4 ℃ refrigerator or cold storage).

(2) The beaker containing the inclusion body renaturation reagent is placed on a magnetic stirrer, a magnetic rotor is added, and the stirring speed is set to be proper (100-.

(3) 1mL of inclusion body (30mg/mL) of m beta 2m dissolved in guanidine hydrochloride is added into a syringe, slowly dropped into the inclusion body renaturation reagent, and slowly stirred for 6-8 h.

(4) 5mg of polypeptide P2 was dissolved in 200. mu.L of DMSO, respectively, and then directly added to the inclusion body renaturation reagent by a pipette.

(5) After stirring slowly for 30min, 3mL of H-2Kb inclusion body (30mg/mL) is added into the syringe, and the action time is prolonged properly by stirring slowly for 8-12H.

II, concentration of the Complex protein

After renaturation is finished, the renaturation solution is carefully transferred to a pressure stirring type concentration cup in a refrigerator or a cold storage at 4 ℃, the pressure is provided by adopting nitrogen, and 120mL of precooled molecular sieve buffer solution is added when the volume of the concentrated solution on the 10kDa ultrafiltration membrane is about 30 mL. Finally, when the solution is concentrated to about 30mL, the solution is transferred to a 50mL centrifuge tube, the precipitate is removed by centrifugation at 12000rpm at 4 ℃, the supernatant is transferred to a 10KD protein concentration tube, and the supernatant is further concentrated to 2-5mL at 2400rpm at 4 ℃. If the renaturation liquid becomes turbid after renaturation, the precipitate can be removed by low-temperature centrifugation, and then the mixture is concentrated by a pressure stirring type concentration cup.

Thirdly, molecular sieve chromatography and ion exchange chromatography identification of the compound protein

Balancing Superdex 20016/60 HiLoad gel chromatographic column with molecular sieve buffer solution, balancing to baseline (generally 1ml/min, about 2 h), centrifuging the concentrated and liquid-changed complex protein sample to remove precipitate (4 deg.C, 12000rpm, 10min), washing the sample pump of Fast Protein Liquid Chromatograph (FPLC) with molecular sieve buffer solution, and collecting supernatant. Running on a chromatographic column at a proper flow rate (generally 1ml/min), detecting the renaturation effect according to the result of molecular sieve chromatography, collecting the target peak protein with the corresponding molecular weight, and identifying by SDS-PAGE. The collection of protein peaks identified as the protein (complex) of interest was further analyzed using anion exchange column Resource Q. The column was equilibrated with ion-exchange solution B (10mM Tris-HCl, 1M NaCl, pH8.0), the column was equilibrated with ion-exchange solution A (10mM Tris-HCl, 10mM NaCl, pH8.0) and loaded, and the sample was suspended on the column and subjected to linear gradient elution with an eluent composed of ion-exchange solution A and ion-exchange solution B for 60 minutes. During the 60 minutes, the volume ratio of the ion-exchange liquid a in the eluent linearly decreased from 100% to 50%, and the volume ratio of the ion-exchange liquid B in the eluent linearly increased from 50% to 100%. The eluted peaks were collected and identified by SDS-PAGE. The complex of H-2Kb protein, mouse m beta 2m protein and polypeptide P2 protein is designated as H-2Kb/Gp40/15-AIF 9.

The results are shown in FIG. 1, wherein A is H-2Kb, m beta 2m and polypeptide P2(H-Kb molecular weight is about 32KD, m beta 2m 10KD, P2 is only a short peptide, negligible) after renaturation. The protein chromatography has 3 main peaks under the ultraviolet absorption condition of 280nm, wherein the peak 1 is a polymer peak of H-2Kb heavy chain, the peak 2 (target protein peak) is a three-molecule complex peak formed by H-2Kb, m beta 2m and polypeptide, the protein at the peak position is collected and analyzed by 12% SDS-PAGE electrophoresis, and the insertion picture is a molecular sieve chromatogram after the polypeptide P2 is renatured and the SDS-PAGE identification result of the collected protein peak. The result shows that the peak 2 at 82mL of elution volume is H-2Kb/P2/m beta 2m complex monomer, the molecular weight of the protein is about 42kDa, and the polypeptide has a certain in vitro affinity with H-2 Kb; the last peak (peak 3) was around 105mL elution volume, being m β 2m monomeric protein.

The target peak was collected and further identified by ion exchange chromatography, and the polypeptide complex was found to dissociate at a conductivity of 12-16mS/cm (see FIG. 1B for the results), indicating that the stability of the polypeptide binding to H-Kb molecules in vitro was low, but subsequent experiments showed that the stability of binding in vitro did not affect the immunogenicity of the polypeptide in vivo.

Example 3 construction of Positive polypeptide tetrameric protein

First, amplification of target Gene

According to the nucleotide sequence of H-2Kb, an upstream primer SU1 and two downstream primers SL-BSP1 and SL-BSP2 are designed, wherein the upstream primer introduces a BamHI enzyme cutting site and the downstream primer XhoI enzyme cutting site. A DNA sequence encoding BSP (substrate peptide of BirA enzyme) 15 amino acids (GSLHHILDAQKMVWNH) in length and a base sequence encoding a Gly-Ser linker were introduced into the H-2Kb downstream primer sequence region using two downstream primers, the information of which is detailed in Table 2. Constructing pET21a/H-2Kb-BSP plasmid, extracting H-2Kb-BSP inclusion body, purifying H-2 Kb-BSP-mbeta 2 m/positive polypeptide compound; biotinylation is carried out on receptor protein by using BirA enzyme, redundant avidin in the biotinylation protein is removed by molecular sieve chromatography, PE-labeled streptavidin is added according to a certain proportion after the concentration of the biotinylation protein is measured, and a tetramer of positive polypeptide is prepared and stored at 4 ℃ for later use.

TABLE 2 primers for amplification of mouse H-2Kb-BSP Gene

Preparation of a Di, H-2Kb-BSP/AIF9 tetramer

1. Preparation of peptide Complex monomers

Monomers (H-2Kb protein, a complex of mouse m.beta.2 m protein and polypeptide protein, H-2Kb complex protein for short) required for preparing tetrameric protein according to the method of example 2, inclusion bodies were extracted and renatured in vitro. The protein complex is purified by molecular sieve chromatography and ion exchange chromatography. Biotinylation of the peptide complexes was performed according to the following system.

The biotinylation was performed by incubation at room temperature overnight.

2. Purification of H-2Kb-BSP/AIF9 after biotinylation

The H-2Kb-BSP/AIF9 complex was centrifuged at 12000rpm for 10min at 4 ℃ to remove the precipitate, and then purified using Superdex (TM) 20016/60 GL gel column to remove unbound D-biotin (D-biotin). The purified biotinylated complex protein was concentrated to 500-.

3. In vitro biotinylation results identification

30. mu.l of streptavidin was divided into two equal portions, 15. mu.l of which was mixed with the concentrated biotinylated complex from the previous step and bound overnight at 4 ℃. The other was used as streptavidin control, and the same mass of biotinylated complex protein was diluted to be used as sample control, and the three samples were analyzed by electrophoresis using 12% SDS-PAGE, the results of which are shown in FIG. 2A. Lanes 1 and 3 are control complexes and streptavidin, lane 2 is biotinylated and streptavidin conjugated complexes, and compared to lanes 1 and 3, lane 2 shows a band greater than 170KD indicating successful biotinylation, and another band around 100KD indicating that streptavidin binds only one complex molecule, also confirming successful biotinylation.

4. Preparation and characterization of tetramers

Dividing the PE-labeled streptavidin required for generating the tetramer into 10 equal parts, adding one part into a purified and concentrated sample of the biotinylated complex, incubating for 30min at 4 ℃ in a dark place, continuously mixing uniformly, and repeating the steps until the reaction is finished. The multimerized reaction product was then added to an ultrafiltration tube with a pore size of 100kDa and centrifuged at 2400g at 4 ℃ to a volume of less than 50. mu.l. The sample was diluted to 4ml with PBS (pH8.0) and centrifuged again to a volume of less than 50. mu.l; after repeated dilution with PBS (pH8.0) four times, PBS (pH8.0) was added again to 4mL, and 8. mu.l of Na-EDTA (stock solution concentration: 0.5mol/L), 2. mu.l of pepsin inhibitor (stock solution concentration: 1mg/mL), and 2. mu.l of leupeptin (stock solution concentration: 2mg/mL) were added to the system. The tetramer concentration was further concentrated to 2-2.5mg/mL and stored at 4 ℃ in the dark. Using the biotinylated complex and the PE-labeled streptavidin as controls, 2. mu.l of the tetramer was subjected to detection by 12% SDS-PAGE (see B in FIG. 2). Lanes 1 and 3 are equal amounts of H-2Kb complex and PE-labeled streptavidin, respectively, and lane 2 is a PE-labeled tetramer molecule; the complexes in lane 2 are significantly reduced and a larger protein band appears than with PE-labeled streptavidin, indicating that the biotinylated H-2Kb-BSP/AIF9 complex binds to the PE-labeled streptavidin to form a tetramer. However, it can be seen that there is still a small amount of free biotinylated complex molecules in the tetramer, excess protein is removed by centrifugation in a 100kDa ultrafiltration centrifuge tube, and after the solution is fully changed with PBS and concentrated 4 times, the concentration is measured at about 2-2.5mg/mL and stored at 4 ℃ in the dark.

Example 4 detection of the function of the specific CTL immune response in experimental animals.

First, the immunity of experimental animals

The viral packaging transfection procedure was as follows:

1. cell plating:

passage 293T/(293FT), before cell spreading, 6 cm Dish was coated with gelatin, 0.5mL gelatin was added, the mixture was placed in an incubator at 37 ℃ for 20min, and the cells were completely aspirated, and then the cells were spread (1 × 10 if transfection was performed the next day)⁶Cells, at this point the cell density is about 50-60%, and the next day 80-90%). The next day, the cells were observed to be 80-90% full and transfection was carried out in 10% serum antibiotic-free DMEM medium before transfection.

2. Transfection procedure

(1) The original medium on the cells was removed 2 hours prior to transfection and replaced with fresh complete medium.

(2) Mu.g plasmid DNA was diluted with 100. mu.L serum-free diluent, and the mixture was mixed well to prepare a DNA diluent.

Note that: serum-free dilutions were recommended using Opti-MEM, serum-free DMEM or 150mM NaCl solution.

(3) Adding directly into the DNA dilutionInto 2 μ L of Neofect^TMAnd (3) slightly and uniformly mixing the transfection reagent, standing for 15-30 minutes at room temperature, and finishing the preparation of the transfection compound.

(4) The transfection complex was added to the cell culture medium and gently mixed.

(5) And continuously culturing for 24-48 hours, collecting cells for identification or adding corresponding antibiotics to screen stable clones.

(6) The virus supernatants were collected 24h and 48h after transfection (with the time after transfection as the start time), filtered through a 0.45um filter or centrifuged at 3000rpm for 5 minutes, collected and stored in a 4 ℃ refrigerator for 14 days at 4 ℃.

The constructed adenovirus vector vaccine is used for immunizing C57BL/6N mice of 6-8 weeks old, and the immunizing dose is 10⁹One virus particle/one virus particle, the immunization method is intramuscular injection of mouse leg. Mice spleen cell immunization was isolated 14 days after immunization and the level of T cell immune response in mice was detected by ELISPOT, intracellular cytokine staining and tetramer techniques.

II, separation of mouse spleen cells

Killing the mice by cervical dislocation method, and putting the mice into 75% alcohol for later use; separating spleen, and slowly peeling spleen with forceps; adding the separated spleen into 1640 culture medium, and grinding with a 40um cell sieve by using an injector head; slowly sucking the grinding fluid into a centrifuge tube at 1500rpm (350-; adding 10ml of cell lysate, performing room temperature lysis for 10min, and centrifuging at 1500rpm for 5 min; washing the cells 1-2 times with 1640 medium to remove red blood cells and connective tissue; after centrifugation, the supernatant was carefully transferred to a new 15ml centrifuge tube, and 10ul of cells were counted (observed as bright circular white dots under a microscope).

Thirdly, detecting mouse IFN-gamma by Enzyme-linked Immunospot Assay (ELISPOT) technology

Taking prepared lymphocyte (about 10)⁶cells), plated on treated ELISPOT plates, added stimulator polypeptide (concentration 10ug/ml) and set positive control (ConA), negative control (no polypeptide added) and blank control (blank medium), respectively;culturing in 37 deg.C CO2 culture medium for 12-48h, wherein ELISPOR plate can not be moved during culture period; after the culture was completed, the cells were discarded, washed 5 times with PBS, and 200ul of PBS was added to each well; adding biotinylated antibody (1ug/ml), and incubating at 37 deg.C for 2 h; washing with PBS for 5 times, adding HRP-Streptavidin, incubating at 37 ℃ for 1h, washing with PBS for 5 times, and adding substrate for color development; after being dried at room temperature, the mixture is read on an ELISpot reading machine, and the result is shown in figure 3, wherein the 4 th polypeptide is P2, the 1-3 are the rest polypeptide results, the 1 amino acid sequence is MIYDYNSGL, the 2 is MIWHKSVNL, the 3 is KMLDKYTRM, the 5 th positive control is, and the 6 th negative control is.

Fourth, intracellular cytokine staining detection mouse in vivo IFN-gamma

Taking separated mouse spleen cells, adding PMA (5ng/ml) and Ionomycin (500ng/ml) into positive control, culturing and incubating the cells at 37 ℃ for 6h, adding polypeptide into the polypeptide group at 10ug/ml and 37 ℃ with 5% CO₂Incubate for 12h and add the same volume of DMSO as the negative control. 5-6h before the culture is finished, 3 mu l of brefeldin A (5mg/ml) is added into every 1ml of cultured cells, the mixture is fully mixed, the transfer of the cell factors is inhibited, and the cells are cultured for 5 h. Cells were harvested by centrifugation of 3minutes at 1500rpm and diluted 1X10 with 2% FBS in PBS⁷cells/mL split into inflow tubes; resuspending with PBS for two times, washing, adding PE-labeled CD8 antibody, incubating for 30min at 4 ℃ in the dark, and washing cells with PBS for one time; adding fixative, fixing cells at room temperature for 10min, and performing FACS heavy suspension; adding a membrane breaking agent permeabilization wash buffer, carrying out a light-shielding reaction at room temperature for 30min, centrifuging at 1000rpm for 5min, centrifuging at 4 ℃, adding a washing solution 1 ml/tube, and washing cells for 2 times; resuspending with 100ul FITC labeled anti-mouse IFN-gamma antibody (recommended dilution factor of antibody: 1:100in 1X Perm/Wash), incubating for 30min, centrifuging at 500g for 3minutes, adding washing solution 1 ml/tube to Wash cells for 2 times, centrifuging at 1000rpm, and centrifuging for 5 min; finally, 300-400. mu.l FACS buffer was used to resuspend the cells for flow detection, as shown in FIG. 4, which is the result of intracellular cytokine staining for the P2 polypeptide. Wherein A is a spot produced in the absence of added polypeptide; b is a spot generated by polypeptide stimulation, and C is a cell surface FITC-labeled CD8 antibody of P2 polypeptide and a PE-labeled IFN-gamma double staining result.

Pentamer and tetramer technology for detecting level of polypeptide-specific T lymphocyte

Take 2 x10⁶Adding FITC labeled anti-mouse CD8 antibody (concentration is 0.5mg/ml) for 1ul into each cell (about 100ul), supplementing 900ul PBS after being protected from light at 4 ℃ for 30min, centrifuging at 1500rpm for 5min, adding FACS liquid for resuspending the cells, adding PE labeled tetramer (1mg/ml) for 2ul, being protected from light at 4 ℃ for 30min, washing for three times at 4 ℃ with PBS, staining, and analyzing the proportion of FITC and PE double positive T cells by a flow cytometer. A blank adjustment group, a FITC-labeled CD8 single-staining group, and a PE-labeled tetramer single-staining group were also set, and the results are shown in FIG. 5.

Spleen cells of the polypeptide immune group and spleen cells of the adjuvant immune group were separated, PE-labeled H-2K B-BSP/AIF9 tetramer prepared above and FITC-labeled anti-mouse CD8 monoclonal antibody were stained in the dark, the ratio of double positive T cells was detected by flow cytometry, and simultaneously, blank cells, FITC and PE-labeled single stain controls were set up, respectively (fig. 5A, 5B, 5C). The results showed that the fraction of specific double positive T cells in the polypeptide immunization group was 0.84% (fig. 5D, 5E), which is significantly higher than the negative control (PBS immunization group) (0.23%) (fig. 5F).

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Zhou teacher's college

<120> cryptosporidium parvum Gp40/15 protein epitope polypeptide and adenovirus vector vaccine thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 324

<212> PRT

<213> Cryptosporidium parvum

<400> 1

Met Arg Leu Ser Leu Ile Ile Val Leu Leu Ser Val Ile Val Ser Ala

1 5 10 15

Val Phe Ser Ala Pro Ala Val Pro Leu Arg Gly Thr Leu Lys Asp Val

20 25 30

Pro Val Glu Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

35 40 45

Ser Ser Ser Ser Ser Thr Ser Thr Val Ala Pro Ala Asn Lys Ala Arg

50 55 60

Thr Gly Glu Asp Ala Glu Gly Ser Gln Asp Ser Ser Gly Thr Glu Ala

65 70 75 80

Ser Gly Ser Gln Gly Ser Glu Glu Glu Gly Ser Glu Asp Asp Gly Gln

85 90 95

Thr Ser Ala Ala Ser Gln Pro Thr Thr Pro Ala Gln Ser Glu Gly Ala

100 105 110

Thr Thr Glu Thr Ile Glu Ala Thr Pro Lys Glu Glu Cys Gly Thr Ser

115 120 125

Phe Val Met Trp Phe Gly Glu Gly Thr Pro Ala Ala Thr Leu Lys Cys

130 135 140

Gly Ala Tyr Thr Ile Val Tyr Ala Pro Ile Lys Asp Gln Thr Asp Pro

145 150 155 160

Ala Pro Arg Tyr Ile Ser Gly Glu Val Thr Ser Val Thr Phe Glu Lys

165 170 175

Ser Tyr Asn Thr Val Lys Ile Lys Val Asn Gly Gln Asp Phe Ser Thr

180 185 190

Leu Ser Ala Asn Ser Ser Ser Pro Thr Glu Asn Gly Gly Ser Ala Gly

195 200 205

Gln Ala Ser Ser Arg Ser Arg Arg Ser Leu Ser Glu Glu Thr Ser Glu

210 215 220

Ala Ala Ala Thr Val Asp Leu Phe Ala Phe Thr Leu Asp Gly Gly Lys

225 230 235 240

Arg Ile Glu Val Ala Val Pro Asn Val Glu Asp Ala Ser Lys Arg Asp

245 250 255

Lys Tyr Ser Leu Val Ala Asp Asp Lys Pro Phe Tyr Thr Gly Ala Asn

260 265 270

Ser Gly Thr Thr Asn Gly Val Tyr Arg Leu Asn Glu Asn Gly Asp Leu

275 280 285

Val Asp Lys Asp Asn Thr Val Leu Leu Lys Asp Ala Gly Ser Ser Ala

290 295 300

Phe Gly Leu Arg Tyr Ile Val Pro Ser Val Phe Ala Ile Phe Ala Ala

305 310 315 320

Leu Phe Val Leu

<210> 2

<211> 975

<212> DNA

<213> Cryptosporidium parvum

<400> 2

atgagattgt cgctcattat cgtattactc tccgttatag tctccgctgt attctcagcc 60

ccagccgttc cactcagagg aactttaaag gatgttcctg ttgagggctc gtcatcgtca 120

tcatcatcat catcatcatc atcatcatca tcatcatcaa catcaaccgt cgcaccagca 180

aataaggcaa gaactggaga agacgcagaa ggcagtcaag attctagtgg tactgaagct 240

tctggtagcc agggttctga agaggaaggt agtgaagacg atggccaaac tagtgctgct 300

tcccaaccca ctactccagc tcaaagtgaa ggcgcaacta ccgaaaccat agaagctact 360

ccaaaagaag aatgcggcac ttcatttgta atgtggttcg gagaaggtac cccagctgcg 420

acattgaagt gtggtgccta cactatcgtc tatgcaccta taaaagacca aacagatccc 480

gcaccaagat atatctctgg tgaagttaca tctgtaacct ttgaaaagag ttataataca 540

gttaaaatca aggttaacgg tcaggatttc agcactctct ctgctaattc aagtagtcca 600

actgaaaatg gcggatctgc gggtcaggct tcatcaagat caagaagatc actctcagag 660

gaaaccagtg aagctgctgc aaccgtcgat ttgtttgcct ttacccttga tggtggtaaa 720

agaattgaag tggctgtacc aaacgtcgaa gatgcatcta aaagagacaa gtacagtttg 780

gttgcagacg ataaaccttt ctataccggc gcaaacagcg gcactaccaa tggtgtctac 840

aggttgaatg agaacggaga cttggttgat aaggacaaca cagttctttt gaaggatgct 900

ggttcctctg cttttggact cagatacatc gttccttccg tttttgcaat ctttgcagcc 960

ttattcgtgt tgtaa 975

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Ile Phe Ala Ala Leu Phe Val Leu

1 5

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctatttttg cggcgctgtt tgtgctg 27