CN116143875A - Cryptosporidium cgd2_3080 oocyst wall outer wall marker protein and application thereof - Google Patents

Cryptosporidium cgd2_3080 oocyst wall outer wall marker protein and application thereof Download PDF

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CN116143875A
CN116143875A CN202210804264.7A CN202210804264A CN116143875A CN 116143875 A CN116143875 A CN 116143875A CN 202210804264 A CN202210804264 A CN 202210804264A CN 116143875 A CN116143875 A CN 116143875A
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朱冠
王东强
尹继刚
武晓东
姜鹏
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Jilin University
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Abstract

The invention discloses a cryptosporidium cgd-3080 oocyst wall outer wall marker protein and application thereof, wherein the base sequence of the protein is shown as a sequence table SEQ ID N0.1; cryptosporidium oocyst wall protein specific polypeptide, the amino sequence of which is: SGSATQGGPSTTEC; an antibody against the oocyst wall outer wall protein of cryptosporidium, which specifically binds to the outer surface of the oocyst wall outer wall of cryptosporidium; an antibody for resisting the specific polypeptide of the oocyst wall of the cryptosporidium, which is an antibody prepared by adopting the protein of the specific polypeptide of the oocyst wall protein of the cryptosporidium; application of anti-cryptosporidium oocyst wall outer wall protein antibody or anti-cryptosporidium oocyst wall protein specific polypeptide antibody in detecting cryptosporidium; the polypeptide rabbit serum antibody has good sensitivity, when the serum is 1: the fluorescence intensity is bright when diluted by 100; in the detection of a sample in the environment, the polypeptide antibody only recognizes cryptosporidium oocysts, does not cross react and has good specificity.

Description

Cryptosporidium cgd2_3080 oocyst wall outer wall marker protein and application thereof
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a cryptosporidium cgd-3080 oocyst wall outer wall marker protein and application thereof.
Background
Cryptosporidium is a genus under the parasite Achillea (Apicomplexa), and includes multiple species that infect humans or animals. Wherein, the Cryptosporidium parvum is co-suffered by human and animalsCryptosporidium parvum) And human Cryptosporidium specific to human bodyCryptosporidium hominis) Is two major pathogens causing severe, even fatal diarrhea caused by cryptosporidiosis worldwide; cryptosporidium parvum can also infect a plurality of economic animals including cattle, goats, sheep and the like, and can cause severe diarrhea and death of young animals. Cryptosporidium is a parasite in the gastrointestinal tract, and is mainly transmitted through the faecal-oral route, and the host is infected after taking water or food and the like polluted by the Cryptosporidium. It is mainly hosted in small intestine epithelial cells, and causes symptoms to the host that vary in magnitude, mainly manifested as moderate to severe diarrhea. Cryptosporidiosis is generally self-limiting in immunocompetent humans and animals; but is a major cause of mortality in immunocompromised hosts (e.g., aids patients). Cryptosporidium oocysts are round or oval in shape, microscopic in size (e.g., cryptosporidium parvum oocysts have a diameter of about 5 microns), and the major structure includes the oocyst wall and 4 sporozoites that it encapsulates, and can survive in the environment for a long period of time (FIG. 1). Oocyst wall structure is specific and can resist conventional sodium hypochlorite disinfection, so that cryptosporidium is a great difficulty in controlling cryptosporidium parvum pollution in foods (such as vegetables, berries and the like) and water samples (such as drinking water, recreational water and the like). No specific medicine and vaccine for cryptosporidium parvum exists so far, which further increases the demands for cryptosporidium parvum control work. At present, the detection method for cryptosporidium parvum in human or animal clinical samples in the environment mainly comprises etiology detection, molecular biology detection and immunological detectionOocyst or worm antigens. The pathogen detection can directly observe undyed or marked oocysts through a microscope, but the method is time-consuming and labor-consuming, has extremely low sensitivity and needs to be operated by a skilled technician; the existing molecular biological diagnosis has higher sensitivity and specificity, but has higher requirements on detection instruments; the immunological detection method has certain sensitivity and specificity, is suitable for clinical examination and environmental sample monitoring, but needs to have an antibody capable of specifically marking the outer wall of the oocyst. In addition, the oocysts of the insect body in the environment are usually highly diluted, the oocysts of partial clinical samples are low, and the detection of the samples needs to be carried out on enrichment and concentration of the oocysts before the diagnosis method is used (a non-specific physical method is that only centrifugal sedimentation is adopted, high-density liquid floating is adopted and centrifugal sedimentation is combined, a specific immunological method is adopted, small particles or small magnetic beads with even chains of antibodies capable of marking the outer wall of the oocysts of the cryptosporidium are used for capturing the oocysts, and then the particles or the magnetic beads are collected through centrifugal sedimentation or magnetic object sedimentation).
The current domestic clinical detection method of Cryptosporidium mainly relies on acid-fast staining and other methods to detect oocysts, and the method has low specificity and poor sensitivity. At present, a diagnosis kit for marking the oocyst wall of cryptosporidium by using a fluorescent antibody is disclosed, wherein the oocyst wall is marked by using an antibody fluorescent prepared by taking an oocyst wall protein with detailed specific genetic information as an antigen (only one protein), and the kit is used for microscopic observation and detection. The antibodies are also used for immunological enrichment of cryptosporidium oocysts in a sample. The detection kit based on the antibody is high in price and not easy to be used on a large scale. And the method for detecting the cryptosporidium in an environmental water sample or a clinical sample by using indirect Immunofluorescence (IFA) is blank in China, mainly because no protein which can be used for researching and developing specific antibodies for marking the oocyst outer wall and is positioned on the oocyst outer wall of the cryptosporidium is still found in the current field. Therefore, the method is very significant in monitoring cryptosporidium in a water sample, detecting and diagnosing clinical cryptosporidium diseases, investigating epidemic diseases of human and animal cryptosporidium infection, preventing and treating human and animal co-suffering cryptosporidium and the like.
Disclosure of Invention
The invention aims to solve the problems and provides a cryptosporidium cgd2_3080 oocyst wall outer wall marker protein and application thereof.
The base sequence of the cryptosporidium cgd-3080 oocyst wall outer wall protein is shown as SEQ ID N0.1 of a sequence table; it is located on the outer surface of the oocyst wall outer wall.
A cryptosporidium cgd2_3080 oocyst wall protein specific polypeptide having the amino sequence: SGSATQGGPSTTEC.
An immunological formulation comprising the cryptosporidium cgd2_3080 oocyst wall protein specific polypeptide of claim 2 coupled to KLH/BSA.
An antibody against cryptosporidium cgd2_3080 oocyst wall outer wall protein, which is characterized in that: an antibody which can be specifically combined with the outer surface of the outer wall of the oocyst wall of the cryptosporidium cgd-3080, and is prepared by using the marker protein of the outer wall of the oocyst wall of the cryptosporidium cgd-3080 or the specific polypeptide of the oocyst wall protein of the cryptosporidium as claimed in claim 2.
The application of the anti-cryptosporidium cgd-3080 oocyst wall outer wall protein antibody in detecting cryptosporidium oocysts.
The Cryptosporidium is oocyst of Cryptosporidium without breaking wall.
The invention provides a cryptosporidium cgd-3080 oocyst wall outer wall marker protein and application thereof, and the base sequence of the protein is shown as a sequence table SEQ ID N0.1; cryptosporidium oocyst wall protein specific polypeptide, the amino sequence of which is: SGSATQGGPSTTEC; an antibody against the oocyst wall outer wall protein of cryptosporidium, which specifically binds to the outer surface of the oocyst wall outer wall of cryptosporidium; an antibody for resisting the specific polypeptide of the oocyst wall of the cryptosporidium, which is an antibody prepared by adopting the protein of the specific polypeptide of the oocyst wall protein of the cryptosporidium; application of anti-cryptosporidium oocyst wall outer wall protein antibody or anti-cryptosporidium oocyst wall protein specific polypeptide antibody in detecting cryptosporidium; the polypeptide rabbit serum antibody has good sensitivity, when the serum is 1: the fluorescence intensity is bright when diluted by 100; in the detection of a sample in the environment, the polypeptide antibody only recognizes cryptosporidium oocysts, does not cross react and has good specificity.
Drawings
FIG. 1 this is a graph of the successful amplification of a fragment of interest for the cgd2_3080 gene;
FIG. 2 the recombinant protein demonstrates that it is an oocyst wall outer wall protein;
FIG. 3 shows a graph of specific polypeptide antibody titer detection;
FIG. 4 shows that the specific polypeptide antibody is an oocyst wall outer wall protein by IFA;
FIG. 5 shows that the specific polypeptide antibody is oocyst wall outer wall protein through an Immune Electron Microscope (IEM);
FIG. 6 detection sensitivity of polypeptide antibodies to different titers;
FIG. 7 is a different sample-specific assay.
Detailed Description
EXAMPLE 1 recombinant protein expression of protein of Cryptosporidium parvum Gene cgd2_3080
1. Screening of Cryptosporidium parvum cgd2_3080 protein recombinant protein
(1) The gene number cgd2_3080 (CpTRAP 10) was searched on the professional website of Cryptosporidium (http:// cryptodb. Org/cryptodb /), and the nucleotide and amino acid sequences thereof were downloaded.
(2) Construction of prokaryotic expression vectors
Aiming at the constructed prokaryotic expression vector:
the nucleotide sequence (SEQ ID N0.1) of the protein (amino acid sequence SEQ ID N0.2) was ligated to expression vector pET-28a to express recombinant protein rCpTRAP10.
Aiming at cgd-3080 base sequence, designing a primer, and selecting proper enzyme cutting sites by utilizing DNAMAN, wherein the selected enzyme cutting sites are as follows: upstream:SalI （GTCGAC）, Xho I(CTCGAA) and blast alignment of primer specificity, primers were designed as follows: F-GCGTCGACATTCTCAATCCTGAAGGTGGG, R-CCGCTCGAGctaACTCATACAGCCTTCTCCTT, amplified fragment size 975 bp. The designed primers were synthesized by Jilin Kumei biosystems.
Extracting Cryptosporidium parvum DNA as a template, adding the primer, and carrying out PCR by using high-fidelity enzyme (Nuo-uzan P515-01), wherein the reaction system is as follows: 2 ⨯ Phanta Max Master Mix premix liquid 25 [ mu ] L, each 2 [ mu ] L of upstream and downstream primers, 1 [ mu ] L of DNA template, 20 [ mu ] L of water and 50 [ mu ] L of total volume; the reaction procedure is: pre-denaturation at 95℃for 3 min; denaturation: 95 ℃,15 s; annealing at 60 deg.c, 30 and s; extension: 72 ℃,60 s; after the PCR product is identified by nucleic acid electrophoresis at 72 ℃ for 5 min and 35 cycles, the gene fragment is recovered by using a gel recovery kit, and then the concentration of the gene fragment is measured for enzyme digestion, and simultaneously, an empty vector pET-28a is prepared.
Double digestion is carried out on the target fragment and the empty vector pET-28a, and according to the specification of enzyme, the digestion system is as follows: 1.μg plasmid;SalI,Xho I each 1 mu L;10 XBuffer 2 [ mu ] L; water was added to 20 μl. Enzyme cutting conditions: the target fragment was recovered by checking the cleavage effect in a 37℃water bath 1h, then concentration was measured, the vector was ligated to the fragment according to the Solution I (Takara) protocol (molar ratio of fragment to vector 5:1) in a 16℃water bath for 30 min, then transferred to BL21 (condonplus) competence, plated on ampicillin-resistant solid medium, PCR identified and sequenced, and the identified correct expression strain was inoculated to the strain containing A + Culturing in liquid culture medium.
2. Expression and purification of recombinant proteins
(1) Screening and optimization of expression conditions
Strains successfully ligated to pET-28a vector (His tag) were streaked and single colonies were picked and cultured in 5 mL liquid medium for 8-10 hours. Then, the bacterial liquid was cultured for about 2 hours at a ratio of 1:100, and when the OD value of 600. 600 nm reached about 0.6, the induced expression was performed. Adding IPTG with the final concentration of 0.1 mmol/L for induction, and setting three different induction temperatures and durations at 16 ℃ and 12 h respectively; 25 ℃,6 h;37 ℃,3 h. And simultaneously, setting up a control group without adding IPTG, and determining the optimal expression condition.
(2) Purification of His-tagged recombinant proteins
1) After proper induction conditions are selected to induce protein expression, bacterial liquid is collected, and centrifugation is carried out at 3500 r/min for 10 min at 4 ℃, and then supernatant is discarded.
2) Adding a proper amount of PBS buffer solution to fully suspend the sediment.
3) The precipitate was vortexed with 20 mL of 10 mM imidazole solution, PMSF (0.5 mmol/L) and Triton X-100 (1%) were added and mixed, the centrifuge tube was placed in an ice-water mixture, the cells were broken with an ultrasonic breaker, and ultrasonic parameters were set: the working time is 15 min, the ultrasonic wave is 3 s, the stop is 3 s, and the power is 160W.
4) The sonicated liquid was centrifuged at 16400 rpm/min at 4℃for 30 min and the supernatant was collected.
5) By His GraviTrap TM The recombinant protein was purified by column and after 20% ethanol in the nickel column was run out, 20 mL of 5 mM imidazole solution was added to equilibrate the column. 10 After the completion of the mM imidazole flow, the lysate supernatant was slowly added, and incubated for about 2 hours, and then allowed to flow through the nickel column slowly. Then 20 mM, 40 mM, 60 mM, 80 mM and 100 mM imidazole are added in sequence for impurity washing. Finally, 500 mM imidazole 1 mL is added, incubated for 10 min, and the effluent is collected and repeated 3 times. The whole process is carried out at 4 ℃.
6) Precooling PBS, dialyzing the protein to remove imidazole, and dialyzing for a period of time not less than 1h to obtain recombinant protein rCpTRAP10. Protein concentration was determined after dialysis and the recombinant protein was identified by SDS-PAGE and Western blot.
EXAMPLE 2 preparation of recombinant protein animal immunization and antibodies and indirect immunofluorescence detection
1. Experimental animal immunization program
The recombinant protein is used for immunizing New Zealand white rabbits. The first immunization was performed by taking 300 μg/vehicle (recombinant protein) and equivalent volume emulsification with Freund's complete adjuvant, and intradermal multi-point injection. Two weeks after the first immunization, the recombinant protein (150. Mu.g/dose) was mixed and emulsified with Freund's incomplete adjuvant in equal volumes for four times. Serum from 14 days before the first immunization and after the fourth immunization was collected from the marginal venous blood, and the serum was isolated for subsequent detection.
2. Indirect immunofluorescence assay
For environmental or clinical samples, removing most bacteria and impurities by using a saturated saline water floating or sucrose floating method, and then performing subsequent tests, wherein the following steps are performed;
(1) Processing the cover glass: the coverslip was treated with 0.1. 0.1 mg/mL polylysine for 30 min, ddH 2 O was rinsed once and air dried at room temperature for 2 hours.
(2) Fixing the sample: the intact cryptosporidium parvum oocysts were fixed with 4% paraformaldehyde for 30 min at room temperature and washed with PBS to remove excess formaldehyde.
(3) Drawing circles and coating samples: drawing a small circle at the center of the processed cover glass by using a grouping pen, taking 30 mu L of sporozoites to drop in the small circle, and placing 1h at room temperature;
(4) Cleaning: sucking the liquid on the cover glass, washing with PBS for 3-4 times and 5 min/time;
(5) An antibody: after 1:50 dilution of antisera with 3% BSA in PBS, 50. Mu.L was added to the coverslip and incubated at room temperature for 1h or overnight at 4 ℃. (note: verify the oocyst wall outer wall protein: antibody incubation for 15 min, and the sample was not permeabilized, and could be stained with green fluorescence (the prepared antibody recognizes the protein), the protein was demonstrated to be oocyst wall outer wall protein, as shown in FIG. 1).
(6) And (2) secondary antibody: after PBS washing, alexa Fluor 488 goat anti-rabit IgG (1:1,000) was added to each well, protected from light at 37℃for 1h.
(7) Nuclear dyeing: washing with PBS, adding DAPI (4', 6-diamidino-2-phenylindole) with a final concentration of 1 mug/mL into each hole, dying the core at room temperature for 5 min in a dark place, and washing with PBS for 3-4 times.
(8) Sealing piece: a drop of anti-fluorescence quencher blocking solution (Beyotime, P0126-5 mL) was dropped, and the film was observed under a fluorescence microscope after the blocking solution was blocked.
As shown in figure 1, the recombinant protein antibodies prove that the protein is the cryptosporidium oocyst wall outer wall protein, and the fluorescence signal is strong, so that the protein has potential establishment value. In order to more exactly confirm that the peptide is oocyst wall outer wall protein and has the greatest effect of improving the sensitivity and the specificity of detection, the peptide sequence with good specificity of 1 segment is designed for further evidence.
EXAMPLE 3 screening of Cryptosporidium parvum cgd2 _3080-specific Polypeptides
(1) Searching the gene number cgd2_3080 on a professional website (http:// cryptoptodb. Org/cryptoptodb /) of cryptosporidium, and downloading the amino acid sequence of the cryptosporidium;
(2) Design of specific polypeptides
Screening for specific polypeptides:
the downloaded amino acid sequence of the gene is input into the website swissmodel. Expasy. Org, the spatial structure is predicted, and a polypeptide fragment of 10-14 amino acids in the Loop region is searched. Then, the B cell epitope is analyzed by using the iedb. Org website, and fragments with higher predicted values are selected. Finally, NCBI is utilized to carry out specificity comparison analysis, and fragments with stronger specificity are selected for synthesis.
(3) In order to enhance the coupling efficiency of the polypeptide to KLH and BSA, a cysteine-containing polypeptide was selectively avoided and a cysteine was artificially added to the polypeptide without cysteine at the N-terminus or C-terminus when designing the polypeptide. The designed polypeptide sequence is as follows: SGSATQGGPSTTEC the polypeptide was synthesized by Shanghai Yao Biotechnology Co.
(4) Coupling of the polypeptide to KLH/BSA 1 mg of KLH/BSA was first dissolved in 200. Mu.L of ddH 2 In O, 200. Mu.g of MBS (m-maleimide benzoyl-N-hydroxysuccinimide) was dissolved in 0.04. 0.04 mL DMF (dimethylformamide) solution, added to the carrier protein solution, mixed for 2 h at RT and dialyzed against PBS overnight. Dissolving 2 mg polypeptide in PBS (phosphate buffer solution) of 0.4 mL, adding the overnight dialyzed mixture into 2 parts of polypeptide solution, respectively, performing RT action of 4 h, dialyzing for 12 h, subpackaging for 20 mu L, and preserving at-20deg.C. The above-mentioned coupled BSA-peptides were identified by SDS-PAGE.
EXAMPLE 4 preparation of animal immunization and polyclonal antibodies and potency determination
1. Experimental animal immunization program
The peptide (SGSATQGGPSTTEC) -KLH which is successfully coupled with the polypeptide can immunize New Zealand white rabbits. The coupled polypeptide (300 mug/dose) is emulsified with Freund's complete adjuvant in equal volume for the first immunization and injected intradermally. Two weeks later, peptide-KLH (150. Mu.g/dose) was mixed and emulsified with Freund's incomplete adjuvant in equal volumes and used as booster four times. Serum from 14 days before the first immunization and after the fourth immunization was collected from the marginal venous blood, and the serum was isolated for detection of antibody titer.
2. Indirect ELISA method for detecting serum antibody titer
The coupled BSA polypeptide is used as a coating antigen, serum before immunization is used as a negative control, serum after 4 times of immunization is used as a primary antibody, goat anti-rabbit IgG (H+L) marked by Alkaline Phosphatase (AP) is used as a secondary antibody, and indirect ELISA detection is carried out. The final concentration of the coating antigen was 5. Mu.g/mL, and the serum was from 1:500 is diluted by a double ratio, the secondary antibody is diluted according to a ratio of 1:20000, a color development liquid is added, and an enzyme-labeled instrument is used for detecting an A405 value.
(1) Coating antigen: diluting 5 mug/mL of peptide-BSA which has been successfully coupled with a Coating Buffer (0.05M carbonate Buffer pH 9.6), 50 mug/well, 37 ℃ after 1 hour, overnight at 4 ℃; or 37 ℃ for two hours, and the next step can be carried out.
(2) Washing the plate: the plates were washed 3-4 times with a wash Buffer (0.05% Tween-20,8g NaCl/L) and 4 min apart with an ELISA plate washer.
(3) Closing: 100 μl/well of Blocking Buffer (3% BSA in 0.05M carbonate Buffer pH 9.6) was added and incubated at 37 ℃ for 1h.
(4) Washing the plate: and (2) the same step.
(5) Incubating primary antibody: the antibodies were diluted with Tween Buffer (PBS solution containing 0.5% BSA and 0.05% Tween-20), with serum before the first immunization as negative control, serum after the last immunization as positive antibody, serum before immunization as negative control, diluted with 1:500,1:1000,1:2000,1:4000,1:8000,1:16000,1:32000 dilution, 50. Mu.L per well, incubated at 37℃for 1h.
(6) Washing the plate: same (2)
(7) Incubating a second enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-rabbit IgG (H+L) was diluted 1:20000 as secondary antibody, 50 μl/well, incubated 1H at 37 ℃.
(8) Washing the plate: same (2)
(9) Color development: preparing a developing solution (PNPP substrate developing) of 1 mg/mL by using a developing solution buffer, developing at 50 mu L/hole at 37 ℃ in a dark place for 10-20 min, detecting an A405 reading value by using an enzyme-labeling instrument, taking a polypeptide coupled with BSA as a detection coating antigen, and detecting the antibody titer of rabbit serum immunized by using KLH-polypeptide by using an ELISA method.
As shown in FIG. 2, after four immunizations of rabbits, the serum effective titer reaches 1:32,000, and the subsequent experiments can be performed.
Example 5 immunoelectron microscope markers
And (3) performing immunoelectron microscope labeling by using the specific polypeptide, and further verifying that the polypeptide is oocyst wall outer wall protein.
1. Preparation of electron microscope samples
(1) Removing the bag: cryptosporidium parvum oocysts were incubated in the decoapsular liquid (0.75% sodium taurocholate in incomplete medium) at 37℃for 1h.
(2) Fixing: 4% paraformaldehyde-0.1% glutaraldehyde fixed 2 h at room temperature, and maleate buffer (containing 0.5 mM maleate) was washed three times for 30 min each.
(3) Block dyeing: the sample was incubated with a maleate buffer containing 0.5% uranyl acetate at 20℃for 30 min and the maleate buffer was washed three times for 15 min each.
(4) Agglutination: blowing the immobilized parasite with completely dissolved 2% low melting point agarose in water bath at 50deg.C, standing at room temperature for 30 min, and cutting Cheng to 0.5 mm 3 Is a small block of (a).
2. Dehydration and penetration of samples
(1) Gradient dehydration of ethanol: 30 Dehydrating with alcohol at 4deg.C for 30 min; dehydrating 1h with 50% alcohol at 4deg.C; 70%,80%,90% alcohol dehydrated at-20 ℃ to 1h each; twice with 100% alcohol 1h.
(2) Pre-soaking: absolute ethyl alcohol: LR-White (2:1) -20deg.C soak 1 h; absolute ethyl alcohol: LR-White (1:1) -20deg.C soak 1 h; absolute ethyl alcohol: LR-White (1:2) -20deg.C soak 1h.
(3) Soaking: the samples were soaked overnight in LR-White at-20 ℃; the sample was saturated 24 h in LR-White at-20 ℃; the sample was saturated with 12 h at-20℃in LR-White.
(4) Polymerization: the samples were embedded using LR-White and placed in gelatin capsules, filled with LR-White, uv polymerized 24 at-15 ℃ h.
3. Immunological markers
(1) Positioning and ultrathin slicing: and (3) selecting a place with rich parasites to carry out ultrathin section with the thickness of 1.5 mu m, fishing the section by using a copper mesh coated with an aromatic film, and fishing the section for a subsequent gold mark by using a nickel mesh coated with the aromatic film after the electronic microscopic examination is qualified.
(2) Closing: the nickel screen was back-buckled on PBS containing 5% skim milk, 0.01% TWEEN 20 (PBS-MT) and incubated at room temperature for 1h.
(3) An antibody: the antibodies were diluted in PBS-MT and washed 2 min X8 times in PBS overnight at 4 ℃.
(4) And (2) secondary antibody: the Goat anti-Rabbit IgG (10 nm Gold) was diluted 1:50 in PBS-MT, incubated at 37℃for 1h, PBS washed 1 min X4 times, deionized water washed 1 min X4 times.
(5) Post-fixing: fixing 2% glutaraldehyde for 10 min at four degrees, and washing with deionized water for 1 min×4 times.
(6) Dyeing: the nickel screen is inserted into 2% uranyl acetate for dyeing for 15 min, and is washed by deionized water for 2 min multiplied by 8 times.
Example 6 Indirect immunofluorescence assay samples
For environmental or clinical samples, removing most bacteria and impurities by using a saturated saline water floating or sucrose floating method, and then performing subsequent tests, wherein the following steps are performed;
(1) Processing the cover glass: the coverslips were treated with 0.1. 0.1 mg/mL polylysine for 30 min, rinsed once with ddH2O, and air dried at room temperature for 2 hours.
(2) Fixing the sample: the whole oocysts were fixed with 4% paraformaldehyde for 30 min at room temperature and excess formaldehyde was washed off with PBS.
(3) Drawing circles and coating samples: drawing a small circle at the center of the processed cover glass by using a grouping pen, taking 30 mu L of sporozoites to drop in the small circle, and standing for 1 hour at room temperature;
(4) Cleaning: sucking the liquid on the cover glass, washing with PBS for 3-4 times and 5 min/time;
(5) An antibody: after 1. Mu.L of the antiserum was diluted 1:50 with PBS containing 3% BSA (bovine serum albumin), 50. Mu.L was added to the coverslip and incubated at room temperature for 1 hour or overnight at 4 ℃. (note: verify for oocyst wall outer wall protein: antibody incubation for 15 min, and sample was not permeabilized and could be stained with fluorescence, the protein was demonstrated to be oocyst wall outer wall protein, as shown in FIG. 1).
(6) And (2) secondary antibody: after PBS washing, alexa Fluor 488 goat anti-rabit IgG (1:1,000) was added to each well, protected from light at 37℃for 1h.
(7) Nuclear dyeing: washing with PBS, adding DAPI (4', 6-diamidino-2-phenylindole) with a final concentration of 1 mug/mL into each hole, dying the core at room temperature for 5 min in a dark place, and washing with PBS for 3-4 times.
(8) Sealing piece: dropping a drop of anti-fluorescence quenching agent, and observing under a fluorescence microscope after the sealing piece is sealed by a sealing piece liquid.
As shown in fig. 4-5, the specific polypeptide antibody can sensitively and specifically recognize the oocyst wall of cryptosporidium, and the polypeptide rabbit serum antibody has good sensitivity, as shown in fig. 6, when serum 1: the fluorescence intensity is very bright when 100 dilutions, and gradually weakens along with continuous dilution of the antibody, and when the serum dilution ratio is listed as 1: 10000. and weak fluorescence is generated, and the experiment shows that the serum antibody sensitivity effect is very good.
(note: verify the oocyst wall outer wall protein: antibody incubation for 15 min, and not permeabilizing the sample (tritol, SDS, etc.), i.e., the antibody only recognizes the oocyst wall outer wall protein, if the fluorescence intensity of the antibody corresponding to the protein is more obvious, it is indicated that the antibody recognizes the protein gastric oocyst wall outer wall protein, as shown in FIG. 1).
In the detection of samples in the environment, as shown in FIG. 7, the polypeptide antibodies only recognize cryptosporidium oocysts (cryptosporidium parvum and cryptosporidium tazii in the upper picture) and do not cross react with other species (escherichia coli, coccidium, etc.), which indicates that the antibody specificity is good.
Therefore, the oocyst wall outer wall protein is selected as a candidate antigen, and has great detection potential for detecting cryptosporidium in environmental water samples or clinical samples as a diagnostic reagent.

Claims (6)

1. The base sequence of the cryptosporidium cgd-3080 oocyst wall outer wall protein is shown as SEQ ID N0.1 of a sequence table; it is located on the outer surface of the oocyst wall outer wall.
2. A cryptosporidium cgd2_3080 oocyst wall protein specific polypeptide having the amino sequence: SGSATQGGPSTTEC.
3. An immunological formulation comprising the cryptosporidium cgd2_3080 oocyst wall protein specific polypeptide of claim 2 coupled to KLH/BSA.
4. An antibody against the oocyst wall outer wall protein of cryptosporidium cgd2_3080, which is characterized in that: an antibody which can be specifically combined with the outer surface of the outer wall of the oocyst wall of the cryptosporidium cgd2_3080, and is prepared by using the marker protein of the outer wall of the oocyst wall of the cryptosporidium cgd2_3080 or the specific polypeptide of the oocyst wall protein of the cryptosporidium as claimed in claim 1.
5. The use of the antibody of the oocyst wall outer wall protein of the anti-cryptosporidium cgd2_3080 of claim 4 for detecting oocysts of cryptosporidium.
6. The use according to claim 5, characterized in that: the Cryptosporidium is oocyst of Cryptosporidium without breaking wall.
CN202210804264.7A 2022-07-09 2022-07-09 Cryptosporidium cgd2_3080 oocyst wall outer wall marker protein and application thereof Pending CN116143875A (en)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
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WO1997008204A1 (en) * 1995-08-30 1997-03-06 Macquarie Research Ltd. Methods for detection of cryptosporidium oocysts
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CN108752423A (en) * 2018-05-25 2018-11-06 吉林大学 A kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application
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