CN111239269A - Method for analyzing coccidian sporozoite surface protein fingerprint spectrum and application of coccidian sporozoite surface protein fingerprint spectrum - Google Patents
Method for analyzing coccidian sporozoite surface protein fingerprint spectrum and application of coccidian sporozoite surface protein fingerprint spectrum Download PDFInfo
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Abstract
The invention relates to a method for analyzing coccidian sporozoite surface protein fingerprint spectrum and application of the coccidian sporozoite surface protein fingerprint spectrum, belonging to the technical field of veterinarians. The method adopts a Biotin labeling reagent Sulfo-NHS-SS-Biotin to label the sporozoite, then uses cell lysate to crack the sporozoite, extracts sporozoite cell membrane protein, purifies the obtained protein by streptavidin-agarose affinity, and then carries out mass spectrum identification analysis, establishes a method for effectively separating the sporozoite surface protein, clarifies the composition of sporozoite surface protein spectrums, and lays a foundation for screening effective surface protein antigens and obtaining vaccine candidate molecule combinations with application potential.
Description
Technical Field
The invention relates to the technical field of veterinarians, in particular to a method for analyzing coccidian sporozoite surface protein fingerprint spectrum and application of the coccidian sporozoite surface protein fingerprint spectrum.
Background
The antigen protein of protozoa mainly comprises cell surface protein (including development stage specific differential protein), invasion related secretory protein, cytoskeletal protein, biochemical metabolic enzyme and the like. The worm surface protein is a key molecule for interacting with host cells, is directly an antigen substance which is exposed to the immune system of a host at the earliest and is most easily recognized by a host pattern recognition receptor. The analysis of surface protein spectra of plasmodium falciparum, trichuris vaginalis, trypanosoma brucei and the like shows that the surface protein not only directly participates in the invasion of the parasitic protozoa and the parasite-host interaction network, but also is a protective antigen with great application prospect. The analysis of the coccidian surface protein spectrum is an effective way and a key step for screening the high-efficiency vaccine candidate antigen molecules based on the coccidian surface protein. Methods for extracting sporozoite surface protein, such as analysis and identification of chicken coccidian sporozoite surface protein spectrum, have not been reported so far.
Disclosure of Invention
The invention aims to provide a method for analyzing coccidian sporozoite surface protein fingerprint and application of the coccidian sporozoite surface protein fingerprint. The invention adopts a mode of integrating three steps of biotin labeling, membrane protein extraction and streptavidin-agarose affinity purification to obtain the sporozoite surface protein, and combines mass spectrometry sequencing and genome data to identify the sporozoite surface protein.
The invention provides a method for analyzing coccidian sporozoite surface protein fingerprint, which comprises the following steps:
1) washing and suspending the sporozoites by using an ice PBS buffer solution to obtain a sporozoite suspension;
2) mixing the sporozoite suspension obtained in the step 1) with Sulfo-NHS-SS-Biotin, oscillating and incubating, and cleaning with ice PBS buffer solution to obtain surface protein biotinylated sporozoites;
3) suspending the surface protein biotinylation sporozoite obtained in the step 2) in Hanks' solution, adding cell lysate, cracking at 4 ℃ for 45-60 min, and centrifuging at 4 ℃ for 15min at 10000g to obtain supernatant; the cell lysate contains 1% of Triton X-114 by mass volume fraction, 10mmol/L of Tris-Cl with pH value of 7.4, 150mmol/L of NaCl, 1mmol/L of EDTA and 10 muL/mL of 100 Xprotease inhibitor storage liquid; the 100 Xprotease inhibitor storage liquid is a solution containing the analgesic, the gastric inhibitory peptidase and the leupeptin with final concentration of 10 mug/mL;
4) preserving the heat of the supernatant obtained in the step 3) at 37 ℃ for 3min, centrifuging the supernatant at 25 ℃ for 1min at 10000g, and collecting a lower phase to obtain hydrophobic phase protein;
5) adding the hydrophobic phase protein obtained in the step 4) into a streptavidin-agarose resin gel filtration column eluted by PBS buffer solution, eluting by using the PBS buffer solution until the light absorption value of the eluent reaches the lowest at 280nm, and eluting by using 6M guanidine hydrochloride solution or boiled 0.4M urea solution containing 2% SDS by mass to obtain biotin-labeled sporozoite surface protein;
6) performing LC-MS mass spectrometry sequencing on the biotin-labeled sporozoite surface protein obtained in the step 5) to obtain sporozoite surface protein group data, and performing bioinformatics analysis by combining with genome data to obtain the coccidian sporozoite surface protein fingerprint.
Preferably, the PBS buffer of step 1), step 2) and step 5) has a pH of 8.0.
Preferably, the number of washing in step 1) and step 2) is 3.
Preferably, in step 2), each ml of the sporozoite suspension is mixed with 80. mu.L of 10mM Sulfo-NHS-SS-Biotin solution in PBS.
Preferably, the shaking incubation in the step 2) is shaking incubation at 4 ℃ for 2 h.
Preferably, the streptavidin-agarose resin gel filtration column in the step 5) is eluted by PBS buffer solution with 5-10 times of column volume.
Preferably, the pH value of the guanidine hydrochloride solution in the step 5) is 1.5-2.0.
Preferably, the streptavidin-agarose resin of step 5) is streptavidin-agarose resin 6 FF.
The invention also provides application of the coccidian sporozoite surface protein fingerprint spectrum prepared by the method in the technical scheme in preparation of vaccines.
The invention provides a method for analyzing coccidian sporozoite surface protein fingerprint. The method adopts a Biotin labeling reagent (Sulfo-NHS-SS-Biotin) to label the sporozoite, then uses a cell lysate to crack the sporozoite, extracts sporozoite cell membrane protein, carries out affinity purification on the obtained protein by utilizing NeutrAvidin Agarose Resin (streptavidin-Agarose), then carries out mass spectrum identification analysis, establishes a method for effectively separating the sporozoite surface protein, clarifies the composition of sporozoite surface protein spectrum, and lays a foundation for screening effective surface protein antigen and obtaining vaccine candidate molecule combination with application potential. The method of the invention can solve the defect that coccidian sporozoite surface protein is difficult to obtain. Test results show that the coccidian sporozoite surface protein is extracted for the first time, the coccidian sporozoite surface protein fingerprint spectrum is analyzed for the first time, the E.tenella sporozoite surface protein spectrum composition is clarified, and a foundation is laid for expressing main surface protein, evaluating the immune effect of each recombinant protein, screening effective surface protein antigen and obtaining a vaccine candidate molecule combination with application potential.
Drawings
FIG. 1 is a flowchart of the coccidian sporozoite surface protein fingerprint analysis provided by the present invention;
FIG. 2 is an electrophoretogram of sporozoite surface protein provided by the present invention.
Detailed Description
The invention provides a method for analyzing coccidian sporozoite surface protein fingerprint, which comprises the following steps:
1) washing and suspending the sporozoites by using an ice PBS buffer solution to obtain a sporozoite suspension;
2) mixing the sporozoite suspension obtained in the step 1) with Sulfo-NHS-SS-Biotin, oscillating and incubating, and cleaning with ice PBS buffer solution to obtain surface protein biotinylated sporozoites;
3) suspending the surface protein biotinylation sporozoite obtained in the step 2) in Hanks' solution, adding cell lysate, cracking at 4 ℃ for 45-60 min, and centrifuging at 4 ℃ for 15min at 10000g to obtain supernatant; the cell lysate contains 1% of Triton X-114 by mass volume fraction, 7.4 Tris-Cl with 10mmol/LpH value, 150mmol/L NaCl, 1mmol/L EDTA and 10 muL/mL 100 Xprotease inhibitor storage liquid; the 100 Xprotease inhibitor storage liquid is a solution containing the analgesic, the gastric inhibitory peptidase and the leupeptin with final concentration of 10 mug/mL;
4) preserving the heat of the supernatant obtained in the step 3) at 37 ℃ for 3min, centrifuging the supernatant at 25 ℃ for 1min at 10000g, and collecting a lower phase to obtain hydrophobic phase protein;
5) adding the hydrophobic phase protein obtained in the step 4) into a streptavidin-agarose resin gel filtration column eluted by PBS buffer solution, eluting by using the PBS buffer solution until the light absorption value of the eluent reaches the lowest at 280nm, and eluting by using 6M guanidine hydrochloride solution or boiled 0.4M urea solution containing 2% SDS by mass to obtain biotin-labeled sporozoite surface protein;
6) performing LC-MS mass spectrometry sequencing on the biotin-labeled sporozoite surface protein obtained in the step 5) to obtain sporozoite surface protein group data, and performing bioinformatics analysis by combining with genome data to obtain the coccidian sporozoite surface protein fingerprint.
The sporozoites were washed and suspended with ice PBS buffer to give a sporozoite suspension. In the present invention, the pH value of the ice PBS buffer is preferably 8.0, and PBS with the pH value of 8.0 can wash the cells to remove the contaminating foreign proteins. In the present invention, the number of washing is preferably 3 times to remove the foreign proteins. The source of the sporozoite is not particularly limited, the sporozoite preferably comprises coccidian sporozoite, the source of the coccidian sporozoite is not particularly limited, chicken source coccidian sporozoite is selected in the specific embodiment of the invention, and specifically, the sporozoite is obtained by obtaining non-sporulated oocysts from chicken and culturing and sporulating the oocysts to obtain sporozoites. The specific culture and purification method of the sporozoites is not particularly limited, and the conventional culture and purification method is adopted.
After the sporozoite suspension is obtained, the sporozoite suspension is mixed with Sulfo-NHS-SS-Biotin, and the mixture is subjected to shaking incubation and washing by using ice PBS buffer solution to obtain the sporozoite with biotinylated surface protein. In the present invention, the sporozoite suspension is preferably mixed with 80. mu.L of 10mM Sulfo-NHS-SS-Biotin solution in PBS per ml. In the present invention, the shaking incubation is preferably a shaking incubation at 4 ℃ for 2 h. In the present invention, the shaking incubation is preferably a gentle shaking incubation. In the present invention, the pH value of the PBS buffer is preferably 8.0, so that Sulfo-NHS-SS-Biotin can be better dissolved and the activity thereof can be maintained. In the present invention, the number of washing is preferably 3 times to remove the unreacted Biotin reagent Sulfo-NHS-SS-Biotin. The source of the Sulfo-NHS-SS-Biotin is not particularly limited, and the Sulfo-NHS-SS-Biotin can be obtained by adopting conventional commercial Sulfo-NHS-SS-Biotin, for example, the Sulfo-NHS-SS-Biotin is purchased from Seimer Feishell science and technology company, and can react with molecules of primary ammonia contained in membrane protein on the surface of cells to form stable aminoacyl bonds so as to introduce Biotin into the protein.
After the surface protein biotinylation sporozoite is obtained, the surface protein biotinylation sporozoite is suspended in Hanks' solution, cell lysate is added, the cell lysate is cracked at 4 ℃ for 45-60 min, and 10000g of the cell lysate is centrifuged at 4 ℃ for 15min to obtain supernatant; the cell lysate contains 1% of Triton X-114 by mass volume fraction, 7.4 Tris-Cl with 10mmol/LpH value, 150mmol/L NaCl, 1mmol/L EDTA and 10 muL/mL 100 Xprotease inhibitor storage liquid; the 100 Xprotease inhibitor storage liquid is a solution comprising the analgesic, pepstatin and leupeptin all at a final concentration of 10. mu.g/mL. The sources of Hanks 'solution are not particularly limited in the present invention and can be obtained by conventional commercial products of Hanks' solution known to those skilled in the art, such as from solibao, china. The preparation method of Hanks' solution (1L) of the present invention is preferably:
KCl 0.40g, NaCl 8.00g and Na are weighed respectively2HPO4·H2O 0.06g/Na2HPO4·12H2O 0.134g、KH2PO40.06g、NaHCO3Dissolving 0.35g of the extract in 800mL of sterilized deionized water, adjusting the pH value to 7.2-7.4, diluting to 1000mL of the extract, filtering and sterilizing the extract by a 0.22 mu m microporous membrane under an aseptic condition, subpackaging and storing at 4 ℃ for later use.
After obtaining the supernatant, the invention keeps the temperature of the supernatant at 37 ℃ for 3min, centrifuges the supernatant at 25 ℃ for 1min at 10000g, and collects the lower phase (detergent phase) to obtain the hydrophobin. All proteins were dissolved in aqueous solution (cell lysate) containing Triton X-114 at 4 ℃ and at temperatures above 20 ℃ in aqueous and detergent phases, where hydrophilic proteins were dissolved in the aqueous phase and hydrophobic membrane proteins were dissolved in the detergent phase, and membrane proteins were extracted using this property. The detergent phase, i.e. the lower phase protein, according to the invention is a hydrophobic protein.
The method comprises the steps of adding hydrophobic phase protein into a streptavidin-agarose resin gel filtration column eluted by PBS buffer solution, eluting by the PBS buffer solution until the light absorption value of an eluent reaches the lowest value at 280nm (eluting biotin-labeled protein which is not combined with streptavidin-agarose resin 6FF by PBS, and when the light absorption value at 280nm is the lowest, indicating that the biotin-labeled protein is fully combined with the streptavidin-agarose resin 6 FF), eluting by 6M guanidine hydrochloride solution or boiled 0.4M urea solution containing 2% of SDS by mass percentage, and obtaining the biotin-labeled sporozoite surface protein. In the present invention, the streptavidin-agarose resin gel filtration column is preferably eluted with 5 to 10 column volumes of PBS buffer. In the invention, the pH value of the guanidine hydrochloride solution is preferably 1.5-2.0. In the present invention, the solvent of the guanidine hydrochloride solution is preferably water, and preferably Na is added2H2PO4And Tris-Cl plays a stabilizing role. The specific component is 100mM Na2H2PO410mM Tris-Cl,6M guanidine hydrochloride, followed by adjusting the pH to 1.5-2.0 with NaOH. In the present invention, the streptavidin-agarose resin is preferably a streptavidin-agarose resin6FF, the source of which is not particularly limited in the present invention, is available from Saimer Feishale technologies. The eluted sample is immediately dialyzed or desalted as necessary to reduce precipitation (due to rapid pH change), or an alkaline buffer such as 1M Tris solution at pH 9.0 may be added for neutralization to finally obtain the sporozoite surface protein.
After the sporozoite surface protein is marked by the biotin, the method performs LC-MS mass spectrometry on the sporozoite surface protein marked by the biotin to obtain sporozoite surface protein data, and performs bioinformatics analysis by combining with genome data to obtain the coccidian sporozoite surface protein fingerprint. The method for LC-MS mass spectrometry operation is not particularly limited in the present invention, and a protein mass spectrometry sequencing method well known to those skilled in the art may be used. The invention identifies transmembrane related protein and secretory protein to determine the composition of surface protein, preferably adopts ToxoDB (http:// ToxoDB. org/toxo /) database to obtain protein sequence, and SignalP3.0 software predicts the existence of signal peptide; TMHMM Server v.2.0, http:// www.cbs.dtu.dk/services/TMHMM-2.0// to predict transmembrane domain status.
The invention also provides application of the coccidian sporozoite surface protein fingerprint spectrum prepared by the method in the technical scheme in preparation of vaccines.
The following embodiments are provided to further describe the method for analyzing the coccidian sporozoite surface protein fingerprint and the application of the coccidian sporozoite surface protein fingerprint in the invention in detail, and the technical solution of the invention includes but is not limited to the following embodiments.
Example 1
An experimental procedure (flow sheet as shown in FIG. 1):
1, sporozoite purification:
the method comprises the steps of passaging chicks to obtain E.tenella non-sporulated oocysts, adding separated fresh oocysts into a 2.5% potassium dichromate solution, sporulating in a constant-temperature incubator at 28 ℃, when about 95% of oocysts appear, showing that the sporulation process is completed, washing and centrifuging for 3 times to completely remove potassium dichromate, acting for 10min by using a 5% sodium hypochlorite solution at 4 ℃, washing and centrifuging by using sterilized water, and obtaining the primarily purified sporulated oocysts. 10mL of 200mm glass beads were added to a 50mL round bottom centrifuge tube, followed by addition of Hanks' solution solubilized coccidian sporulated oocysts, followed by 5min of beating with wrist force, followed by microscopic observation, and stopping beating if more than 95% of the sporulated oocysts appear to de-capsulate. Preparing 0.25% of trypsin and 0.75% of deoxycholate by using Hanks' solution (pH 7.2-7.4), then incubating in an incubator at 41 ℃, digesting the de-capsulated coccidian beaten by glass beads for 1-2 hours, and detecting whether more sporozoites are released. The digested product was then centrifuged to obtain a pellet, which was resuspended in Hanks' solution, followed by 3500rpm centrifugation for 10min, washing to remove trypsin and deoxycholate, 3 times, and then the resulting cyst-free sporozoites were resuspended in Hanks solution to purify the sporozoites.
The G3 sand core funnel is connected with the rubber plug, vaseline is adopted for pre-lubrication before connection, and then the G3 sand core funnel and rubber plug combination device is connected with a filter bottle with an upper nozzle through the rubber plug to form a whole. Connecting a plastic tube to a filter flask with an upper nozzle, connecting the other side of the filter flask to a vacuum pump, wherein the pressure of the vacuum pump is about 10Mpa, cleaning a G3 sand core funnel by using Hanks 'solution, adding a sporozoite suspension, carrying out suction filtration, centrifuging the liquid obtained by suction filtration to obtain a precipitate, adding Hanks' solution into the precipitate, carrying out heavy suspension, and centrifuging to obtain purified sporozoites.
2 biotinylation of the sporozoite surface protein:
(1) the cells were washed 3 times with ice PBS buffer (pH8.0) to remove contaminating proteins.
(2) The floating spores were suspended in PBS buffer.
(3) To each ml of PBS buffer, 80. mu.L of 10mM Sulfo-NHS-SS-Biotin was added.
(4) Incubate at 4 ℃ with gentle shaking for 2 hours.
(5) The sporozoites were washed 3 times with ice PBS (ph8.0) to remove unreacted biotin reagent.
3, extracting sporozoite surface membrane protein:
(1) suspending biotinylated sporozoites in 0.5mL Hanks' solution, adding cell lysate (1% (w/v) Triton X-114, 10mmol/L Tris & Cl pH 7.4, 150mmol/L NaCl, 1mmol/L EDTA, 10 uL/mL 100 Xproteinase inhibitor storage liquid, wherein the final concentrations of the anti-pain agent, the pepstatin and the leupeptin are all 10 ug/mL), placing the mixture at 4 ℃ for 45-60 min to lyse cells, placing the cell lysate in a microfuge tube, and centrifuging the solution at 10,000g and 4 ℃ for 15 min.
(2) The supernatant was collected into another microfuge tube and incubated at 37 ℃ for 3 min.
(3)10000g were centrifuged at room temperature (25 ℃) for 1min and the lower hydrophobic phase was transferred to another microfuge tube.
(4) 100 μ L of cell lysate was added to the hydrophobic phase of the above experiment and steps 2 and 3 and 4 were repeated to remove the hydrophilic protein residues in the hydrophobic phase to obtain a more pure hydrophobic protein. The lower phase obtained is a hydrophobic protein.
4NeutrAvidin Agarose Resin (streptavidin-Sepharose) affinity chromatography purification of biotinylated proteins:
(1) adding streptavidin-agarose resin 6FF into a gel filtration column, eluting with PBS (5-10 times of column volume), and adding the hydrophobic phase protein obtained in the above step into a column bed.
(2) Elution was performed with PBS until the absorbance at 280nm was the lowest (a characteristic absorption peak at 280nm wavelength due to tyrosine and tryptophan containing conjugated double bonds in the protein molecule. in this wavelength range, A280 of the protein is proportional to its concentration. elution with PBS of the biotin-labeled protein which is not bound to the streptavidin-agarose resin 6 FF. when the absorbance at 280nm is the lowest, it indicates that the biotin-labeled protein is sufficiently bound to the streptavidin-agarose resin 6 FF).
(3) The biotin-labeled protein is eluted by using a 6M guanidine hydrochloride solution (pH value is 1.5-2.0) or a 0.4M urea solution containing boiled 2% SDS.
(4) Immediately dialyzing or desalting the eluted sample to finally obtain the sporozoite surface protein.
The invention dilutes the sporozoite surface protein by 5-10 times, and performs SDS-PAGE electrophoresis (see figure 2, the sporozoite surface protein electrophoresis chart, the leftmost strip is marker, and the right three are sporozoite surface proteins extracted from different batches).
5, protein reduction alkylation and enzymolysis:
(1) after protein quantification, 20 mu g of each sample is taken, precooled acetone with 5 times of volume is adopted for precipitation, the mixture is placed at the temperature of minus 20 ℃ for 1h to fully precipitate the protein, then the mixture is centrifuged at the temperature of 4 ℃ and the speed of 12,000rpm for 10min, and the precipitate is taken and is frozen and dried in vacuum.
(2) The protein precipitate was dissolved sufficiently in 10. mu.L of the protein resuspension, and 40. mu.L of a protein reducing solution (50 mM ammonium bicarbonate solution in water containing 10mM dithiothreitol) was added thereto and reacted at 37 ℃ for 1 hour.
(3) Adding 40 μ L protein alkylation solution (50 mM ammonium bicarbonate aqueous solution containing 50mM indole-3-acetic acid), reacting at room temperature in dark for 10min, adding the protein solution after reductive alkylation into 10kDa centrifugal ultrafiltration tube, centrifuging at 12,000rpm for 20min, and discarding the bottom solution of the collection tube.
(4) Adding 150 μ L of 50mM ammonium bicarbonate solution, centrifuging at 12,000rpm for 20min, discarding the bottom solution of the collection tube, repeating for 3 times, replacing the collection tube, adding 100 μ L of sequencing-grade trypsin solution with concentration of 5 ng/. mu.L into the ultrafiltration tube, and reacting at 37 deg.C for 12 h.
(5) Centrifuging at 12,000rpm for 10min, collecting peptide fragments after enzymolysis, adding 50 μ L of 50mM ammonium bicarbonate aqueous solution into ultrafiltration tube, centrifuging at 12,000rpm for 10min, collecting tube bottom solution, mixing with the previous solution, and lyophilizing.
6 mass spectrum operation and database retrieval:
(1) the lyophilized polypeptide sample was redissolved in Nano-HPLC buffer A (0.1% formic acid-water solution) and separated using Nano-HPLC liquid phase system EASY-nLC 1000. Liquid phase A is 0.1% formic acid-water solution, high performance chromatography mobile phase B: 0.1% formic acid-acetonitrile solution, equilibrate the chromatographic trapping column with 100% solution A, wash the sample room once with a blank solvent mobile phase gradient for 30 min.
(2) The enzymatic products were separated by capillary HPLC and analyzed by mass spectrometry using an orbital hydrazine mass spectrometer (LTQ orbitrapVelos Pro). Analysis duration: 105min, detection mode: positive ion, spray voltage: 1.8kV, ion-transport capillary temperature: 250 ℃, corrected by standard calibration solution before use, parent ion scan range: 350-1800m/z, the mass spectrum scanning mode is an Information-Dependent acquisition working mode (IDA), the strongest 15 fragment patterns (MS2 scan) are acquired after each full scan (full scan), and the fragmentation mode is as follows: collision-induced dissociation (CID), normalized energy 35%, q value 0.25, activation time: 30ms, dynamic exclusion time: for 30 s. The resolution of MS1 was 60,000 at M/Z400, and MS2 was mass resolved in the ion trap. The primary mass spectrum is acquired in a profile mode, and the secondary mass spectrum is acquired in a centroid mode to reduce the size of a data file.
(3) Data processing is carried out by adopting Mascot 2.3 software (Matrix Science), the database adopts an Eimeria database, the enzyme is trypsin, the allowed maximum leaky cleavage site is 2, and the fixed modification is urea methylation (C); the variable modifications are: acetylation (N-terminal protein), deamidation (NQ), dioxygenation (W), and oxidation (M); MSMS tolerance was ± 0.15kDa, protein score c.i.% greater than 95% was successful identification.
The second experiment result is as follows: a number of surface proteins were identified, including the classical ion channel protein ABC transporter, and the like. The proteins are specifically described in table 1:
TABLE 1 identification of surface proteins
The results showed that sporozoites were present with ATP-citrate synthase, predicted cation transport atpase, single-stranded DNA binding protein-related protein, putative protein of SPX domain, ABC transporter, putative peroxidase membrane protein, phosphodiesterase, BT 1-containing transmembrane domain protein, putative Ctr-containing copper ion transporter, putative trypsin, putative ribosomal protein L7, and the like.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for analyzing coccidian sporozoite surface protein fingerprint comprises the following steps:
1) washing and suspending the sporozoites by using an ice PBS buffer solution to obtain a sporozoite suspension;
2) mixing the sporozoite suspension obtained in the step 1) with Sulfo-NHS-SS-Biotin, oscillating and incubating, and cleaning with ice PBS buffer solution to obtain surface protein biotinylated sporozoites;
3) suspending the surface protein biotinylation sporozoite obtained in the step 2) in Hanks' solution, adding cell lysate, cracking at 4 ℃ for 45-60 min, and centrifuging at 4 ℃ for 15min at 10000g to obtain supernatant; the cell lysate contains TritonX-114 with the mass volume fraction of 1%, Tris & Cl with the value of 10mmol/LpH of 7.4, 150mmol/L NaCl, 1mmol/L EDTA and 10 muL/mL 100 Xprotease inhibitor storage liquid; the 100 Xprotease inhibitor storage liquid is a solution containing the analgesic, the gastric inhibitory peptidase and the leupeptin with final concentration of 10 mug/mL;
4) preserving the heat of the supernatant obtained in the step 3) at 37 ℃ for 3min, centrifuging the supernatant at 25 ℃ for 1min at 10000g, and collecting a lower phase to obtain hydrophobic phase protein;
5) adding the hydrophobic phase protein obtained in the step 4) into a streptavidin-agarose resin gel filtration column eluted by PBS buffer solution, eluting by using the PBS buffer solution until the light absorption value of the eluent reaches the lowest at 280nm, and eluting by using 6M guanidine hydrochloride solution or boiled 0.4M urea solution containing 2% SDS by mass to obtain biotin-labeled sporozoite surface protein;
6) performing LC-MS mass spectrometry sequencing on the biotin-labeled sporozoite surface protein obtained in the step 5) to obtain sporozoite surface protein group data, and performing bioinformatics analysis by combining with genome data to obtain the coccidian sporozoite surface protein fingerprint.
2. The method of claim 1, wherein the PBS buffer of step 1), step 2), and step 5) has a pH of 8.0.
3. The method of claim 1, wherein the number of washes of step 1) and step 2) is 3.
4. The method as claimed in claim 1, wherein in step 2), each ml of the sporozoite suspension is mixed with 80 μ L of 10mM Sulfo-NHS-SS-Biotin solution, and the solvent of the Sulfo-NHS-SS-Biotin solution is PBS.
5. The method according to claim 1, wherein the shaking incubation of step 2) is a shaking incubation at 4 ℃ for 2 h.
6. The method of claim 1, wherein the streptavidin-agarose resin gel filtration column of step 5) is eluted with 5 to 10 column volumes of PBS buffer.
7. The method according to claim 1, wherein the pH value of the guanidine hydrochloride solution in the step 5) is 1.5-2.0.
8. The method according to claim 1, wherein the streptavidin-agarose resin of step 5) is streptavidin-agarose resin 6 FF.
9. Use of coccidian sporozoite surface protein fingerprint prepared by the method of any one of claims 1-8 in the preparation of vaccines.
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