CN113896769B - Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody - Google Patents
Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
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Abstract
A preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody. The amino acid sequence of the C-terminal specific polypeptide is as follows: GVSNIRTLDTPFRKNC. The preparation of the anti-human sperm membrane protein SPAG8 antibody comprises the following steps: (1) analysis and design of specific extracellular epitopes; (2) specific artificial polypeptide synthesis; (3) cross-linking the synthetic polypeptide with a carrier protein; (4) preparing a rabbit polyclonal antibody; and (5) collecting, separating and purifying to obtain the antibody. The specific anti-human sperm membrane protein SPAG8 antibody prepared by the invention has high titer, strong affinity and good specificity, can be specifically combined with the human sperm membrane protein SPAG8 epitope with natural activity in vivo and in vitro, and can be used for fluorescent immunity and enzyme-linked immunosorbent assay. The antibody not only provides an important tool for researching the characteristics, functions, expression profiles and related diseases of the human sperm membrane protein SPAG8, but also has application prospects in the fields of targeted tracking, bio-pharmacy, accurate treatment and the like.
Description
Technical Field
The invention relates to a polypeptide and a preparation method of an antibody thereof, wherein the antibody is an important tool for basic research of human sperm membrane protein SPAG8 protein, and has good application prospect in the fields of targeted tracking, bio-pharmacy, accurate treatment and the like.
Background
The number of sterile patients in China is up to 5000 thousands, wherein 10-30% of men activate an autoimmune system due to specific sperm surface membrane antigens, anti-sperm antibodies (AsAb) are generated in vivo, reproductive physiological processes such as sperm movement interference, sperm direct or indirect killing, sperm fusion interference and the like are caused, male sterility is initiated, and the action mechanism of male immune sterility caused by the AsAb is not clear due to the lack of research on sperm membrane protein specific antigen sites and antibodies, so that the interdisciplinary application of technologies such as molecular biology, bioinformatics, high-throughput sequencing and the like further reveals the action mechanism of specific antigen sites of sperm surface membrane proteins of the top body region, neck and tail of sperms in the process of amphiprotic reproduction, and a series of scientific problems such as a specific immune sterility targeted therapy method are established, so that the method is a great demand for promoting healthy fertility, population safety.
(1) SPAG8 function: the human sperm membrane protein SPAG8 (spimm-associated antigen, SPAG 8) gene is located on the 9p13.3 DNA antisense strand, 4,488bp in length, with 9 exons, and two mRNA subtypes have been found: NM_001039592.1 and NM_172312.1.SPAG8 protein products 426 amino acids, molecular weight 44,819Da, divided into 2 subtypes, single transmembrane type II membrane protein and secreted protein, respectively. SPAG8 proteins are widely distributed in the cytoplasm, nucleus, vesicle, and acrosome, and in the cytoskeleton, microtubule tissue center, and mitotic spindle. In mature sperm cells, the distribution was detected in the acrosome region of the head and in the middle part of the tail.
(2) Information on SPAG8 antibody product: polyclonal antibodies to the SPAG8 protein are available on the market, but these antibodies differ in their epitopes and can only be detected in vitro.
(3) SPAG8 and disease: SPAG8 is distributed in the nucleus and cytoplasm in sperm cells and round sperm cells, whereas in deformed sperm cells, it is expressed only in the cytoplasm and not in the nucleus. Mitotic cycle, SPAG8 is localized in the microtubule tissue center (MTOC). Mid-term extension to spindle microtubules; later detection can be performed in the star microtubules and the middle area; the end stage remains in the middle zone. After cell division, the cells return to MTOC, and the protein encoded by the gene is located on the surface of sperm. In spermatogenesis, SPAG8 acts by enhancing tau binding coactivator FHL5 with the cyclic adenosine monophosphate response element regulator subtype and enhancing FHL5 transcriptional activation of tau, furthermore SPAG8 is involved in the acrosome response and in the binding of sperm to the zona pellucida, and by modulating activation of CDK1 required for mitosis to delay entry into the split phase to regulate G2/M progression, SPAG8 may also influence fertility by interacting with RANBP9 that acts in microtubule formation.
Disclosure of Invention
The invention aims at overcoming the defects of an AsAb preparation method for researching an immune sterility mechanism and provides a preparation method of human sperm membrane protein SPAG8 polypeptide and antibody. Different from in vitro experimental detection antibodies, the anti-SPAG 8 antibody prepared by the polypeptide can specifically identify natural human SPAG8 protein in tissues or cells through immunofluorescence and enzyme-linked immunosorbent assay on the one hand, and can conduct research on immune sterility action mechanism mediated by anti-sperm antibody by means of specific surface antigen sites of SPAG8 on the other hand, and can also conduct research on aspects of targeted tracing, bio-pharmacy, accurate treatment and the like.
The technical proposal of the invention
The human sperm membrane protein SPAG8 specific polypeptide has antigen binding sites distributed widely in human sperm neck, tail middle section and tail outer membrane, and has polypeptide sequence of 422 th to 437 th amino acid peptide in human sperm membrane protein SPAG8 amino acid sequence, and the polypeptide has amino acid sequence: GVSNIRTLDTPFRKNC.
The invention also provides a preparation method of the antibody for resisting the human sperm membrane protein SPAG8, which comprises the following steps:
step 1, analyzing and designing specific antigen epitope of human sperm membrane protein SPAG 8;
the amino acid sequence of the protein is subjected to antigen epitope analysis by utilizing biological information software, and indexes such as transmembrane fragments, polypeptide activity, hydrophilicity, antigenicity and the like are mainly evaluated.
Step 1.1 on-line prediction of transmembrane region of Membrane protein Using TMHMM
Logging in a TMHMM main page, submitting a FASTA format SPAG8 protein sequence in a popup box of a 'select file' or pasting the sequence in a text box under 'OR by pasting sequence(s) in FASTA format'; out formats have three options, respectively, graphical output (existence); text output (no graphics); displaying proteins line by line (One line protein), and displaying a default option graphically; after the adjustment is finished, a submit key is pressed to submit and check the prediction analysis result.
Step 1.2 predictive analysis of Signal peptides on Membrane proteins Using SignalP 4.1Server
Logging in a SignalP 4.1Server main page, submitting a FASTA format protein sequence in a pop-up box of a 'selected file' or pasting the sequence in a text box below; parameter setting organosm group: selecting Eukaryotes; d-cutoff values: selecting Default (optimized for correlation); graphics output: selecting PNG (inline); output format: a Standard; method selection Input sequences may include TM regions. And after the parameter setting is finished, pressing a submit key to submit and check a prediction analysis result.
Step 1.3 predictive analysis of hydrophobicity of Membrane proteins Using Expasy-ProtScale
Logging in an ExPASY-ProtScale main interface, and inputting a protein sequence ID number in a text box of 'Enter a UniProtKB/Swiss-Prot or UniProtKB/TrEMBL accession number (AC)'; or the FASTA format protein sequence is stuck to a text box of Or you can paste your own sequence in the box below; and selecting default setting, submitting and checking a prediction analysis result according to the 'subset' after the setting is finished.
Step 2, synthesizing a human sperm membrane protein SPAG8 specific polypeptide antigen and coupling and purifying a carrier protein;
step 2.1 Synthesis of SPAG8 Artificial polypeptide
And synthesizing and purifying by using an automatic polypeptide synthesizer, and identifying the purity by using mass spectrometry and high performance liquid chromatography, wherein the purity is more than 95%.
Step 2.2 Synthesis of polypeptide-KLH by EDC coupling
KLH was prepared as a 10mg/ml solution, the synthetic polypeptide of step 2.1 was dissolved in 2.5ml of Imject@EDC Conjugation Bufjfer at a concentration of 0.4mg/ml, the polypeptide solution was added dropwise to the carrier protein solution, 10mg/ml of EDC solution was prepared with ultrapure water, 250ul of this solution was immediately added to the mcKLH peptide solution, and after 2 hours of reaction at room temperature, a synthetic polypeptide-KLH complex was obtained and the residue of EDC was removed.
Step 2.3, purification of the synthesized polypeptide-KLH Complex
60ml of ultra-pure water degassed by ultrasonic vibration is added into a Purification Buffer Salts bottle to dissolve the content, a desalting column is used for filtering every 0.5ml of sample, 0.5ml of synthesized polypeptide-KLH complex is gently dripped into 25ml of Purification Buffer Salts eluting column, standing is carried out for 2 minutes, 5ml of purification buffer is taken for eluting the column, the absorbance of the eluent is detected at 280nm by ultraviolet-visible absorption spectrometry, and the content of the polypeptide-KLH complex is calculated according to the absorption peak. If the immunogen needs to be stored for a longer period of time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing an artificial immunogen immune animal;
step 3.1, purchase 4 New Zealand white rabbits with SPF grade and weight of 2.0 KG. The normal serum was prepared by drawing blood from the ear vein of animals 0.5ml before the immunization experiment;
step 3.2, antigen is encapsulated with adjuvant: before each immunization, 400-600ug of the polypeptide-KLH complex is sucked into a 2.5ml sterile syringe, the other 2.5ml syringe is used for sucking equal amount of complete Freund's adjuvant or Freund's incomplete adjuvant, the two syringes are connected by a sterile plastic hose and repeatedly pumped until the complete emulsification is achieved (one drop is not diffused in cold water, namely, the complete emulsification degree of water-in-oil is achieved), and the polypeptide-KLH complex can be used for immunization.
Step 3.3, animal sensitization: 400mg of polypeptide-KLH complex and complete Freund's adjuvant in a volume ratio of 1: step 2, mixing, namely performing animal sensitization after fully mixing, and injecting the mixture into the skin by adopting a multipoint method (about 200ul per point) on the trunk, neck and back of the animal in order to increase the sensitization effect;
step 3.4, cross-emulsifying antigen with Freund's incomplete adjuvant and Freund's complete adjuvant at the same antigen dose and boosting once a week, emulsification method was as before. The injection is respectively carried out at the subcutaneous parts of the back, the subcutaneous parts of the abdomen, the inguinal canal, the popliteal canal, the sole and other parts of the rabbit. During injection, the rabbit is fixed by the rabbit box, the skin is lifted by the left hand, the syringe is held by the right hand, the angle between the needle head and the skin is adjusted to be about 15 degrees, the needle head is injected into the skin for 1-2cm and then lifted upwards to prevent the needle head from penetrating into the muscle, and about 200ul of the needle head is injected at each point.
Step 3.5, specific immunization protocol is shown below, for a total of 10 weeks. Each rabbit was boosted twice by the ear vein at weeks 7 and 9. The Elsia experiments were performed at weeks 4,8, 9 and 10 to detect antibody titers and specificity, respectively.
Step 4, collecting antibody serum;
and 4.1, fixing four limbs of the rabbit in a supine position by using ropes, wherein the two upper limbs are fixed at the back of the head in a crossed manner, tying the upper jaw and the incisors of the rabbit by using the ropes, and pulling the rabbit backwards to fix the rabbit head on the two upper limbs in a homeopathic manner.
Step 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck along the middle of the neck from the upper fossa sternum to the lower jaw for about 15cm, carefully separating subcutaneous tissues along the trachea after finding out the trachea, and obtaining the mastoid muscle from the distal end to the throat and from the proximal end to the sternoclavicular muscle.
Step 4.3, the beating carotid artery is available below the trachea, the bilateral carotid arteries are carefully isolated and sufficiently dissociated.
Step 4.4, sleeving two black wires into one artery and separating the two black wires (one at the distal end and one at the proximal end), ligating the distal end of the artery by using the wires, clamping the proximal end of the artery by using an arterial clamp, cutting a small opening on the artery wall between the wires and the arterial clamp by using ophthalmic scissors, rapidly inserting a prefabricated thin plastic hose, and rapidly fixing the hose and the artery by using the proximal wire so as to prevent the hose from falling out and leaking blood.
Step 4.5, lightly loosening the arterial clamp, obliquely placing the arterial clamp by using a 50ml centrifuge tube, and increasing the blood discharge amount by treating the carotid artery at the other side in the same way until no blood drips out, and extruding the heart when the blood discharge amount is slow so as to increase the blood discharge amount.
Step 4.6, isolation and preservation of rabbit antisera rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first aspiration of serum, the serum was aspirated a second time by centrifugation at 12000 rpm at 4 degrees celsius for 15 minutes. Finally, about 50ml of SPAG8 antibody serum is obtained, and after being split-packed by a 15ml centrifuge tube, naN3 with the volume of 0.01 percent is added respectively, and the mixture is placed into a refrigerator with the temperature of minus 20 ℃ for storage.
Step 5, separating and purifying the anti-human sperm membrane protein SPAG8IgG antibody by a saturated ammonium sulfate method;
step 5.1, 3ml of rabbit antiserum was transferred into a 15ml centrifuge tube, 3ml of 0.01M pH7.4PBS was gradually added at 4℃and 6ml of saturated ammonium sulfate solution at pH7.0 was gradually added dropwise with slow shaking.
Step 5.2 when the ammonium sulphate solution reached 50% saturation, the solution was left at 4℃overnight.
Step 5.3, centrifuging the solution at 4 ℃ for 20min at 10000 revolutions per minute, and removing the supernatant to obtain the globulin precipitate.
Step 5.4, after dissolving the precipitate in 3ml of 0.01M PH7.4PBS at 4 ℃, 1.5ml of saturated ammonium sulfate solution (33% saturation of the ammonium sulfate solution) was added dropwise, and the precipitate was taken for 30min. The procedure was repeated twice.
Step 5.5, centrifuging at 4 ℃ for 10min at 10000 revolutions per minute, and discarding the supernatant to obtain antibody IgG precipitate.
Step 5.6, the supernatant was discarded, 3ml of 0.01M PH7.4PBS was added to dissolve the antibody IgG pellet, and the pellet was filled into dialysis bags.
Step 5.7, dialyzing the dialysis bag in 0.01M PH7.4PBS solution at 4deg.C. The liquid is changed for several times until the external liquid for dialysis has no yellow change. 8) And taking a sample in the dialysis bag, drying in vacuum, taking a small amount of sample, diluting by a proper multiple, and measuring the protein content.
Step 6, identifying the binding titer of the serum antibody and the natural human SPAG8 protein by an ELISA;
step 6.1 preparation of soluble antigen of human sperm Membrane protein
After 3-5d of forbidden appetite of healthy men, the masturbation method takes semen. After the sperm is liquefied in a water bath at 37 ℃, carrying out routine semen analysis, and collecting semen with the sperm survival rate of more than 60 percent, the sperm motility a grade of more than 25 percent, or the sperm with the sperm motility a grade of more than 50 percent for standby; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form two layers of Percoll density gradient separation columns; 4ml of semen to be used was carefully added to the centrifuge tube and a clear interface was seen. 3000 rpm, centrifuging for 15min, carefully sucking out sperm layer, adding into 2ml PBS, centrifuging at 4deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Clean sperm cell pellet was obtained and thoroughly mixed with about 3-5ml PBS and transferred to a 15ml sterile centrifuge tube to obtain sperm cell suspension.
Step 6.1.2, breaking up sperm cell membranes by using an ultrasonic cytoclasis instrument, and fully exposing antigenic sites by membrane proteins. The method comprises the following steps: 3-5ml of a 15ml centrifuge tube containing sperm cells was placed in crushed ice, the top cap was opened, an ultrasonic cytobreaker probe was placed in the solution, the intensity was adjusted to 70%, and the breaking time was 6min (breaking 5s, suspending 5s to prevent temperature superantigen denaturation and foam generation).
Step 6.1.3, centrifuging the solution (4 ℃ C., 12000 r/min), discarding the precipitate, collecting supernatant (sperm-soluble membrane protein antigen), and preserving at 4 ℃ C. Repeating the steps, collecting the soluble antigens obtained in the steps and storing the soluble antigens in a refrigerator at the temperature of-80 ℃.
Step 6.2, coating, namely diluting the humanized sperm membrane protein antigen to 50ug/ml, dripping the diluted humanized sperm membrane protein antigen into a plurality of small holes of a 96-well plate, 100 mu L/hole and standing at 4 ℃ overnight.
Step 6.3, sealing: the soluble antigen in the wells was discarded, and 150. Mu.L/well of 5% nonfat dry milk (blocking solution) was added and incubated at 37℃for 40min.
Step 6.4, washing, namely adding 200 ul/hole of washing liquid (PBST), washing three times, and standing for 3min each time.
Step 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of serum antibodies to be tested with different dilutions, and standing at 37 ℃ for 40min.
And 6.6, washing, namely adding PBST into the small holes, standing for 3min, discarding the washing liquid in the holes, repeating for three times, and finally fully throwing away the washing liquid in the holes.
Step 6.7, adding enzyme-labeled antibody (secondary antibody) by diluting HRP-labeled goat anti-rabbit secondary antibody by 1:10000, adding 100 μl/well, and standing at 37deg.C for 40min.
Step 6.8, wash with PBST three times, 3min each time.
And 6.9, developing, namely adding 100 mu L/hole of TMB developing solution, and standing for 15min at normal temperature in a dark place.
Step 6.10, stop solution, 1M H2SO4, 50. Mu.L/well.
Step 6.11, measuring, namely measuring the OD 490nm value by using an enzyme-labeled instrument.
Step 7, positioning SPAG8 protein distribution on the surface of the sperm by using cellular immunofluorescence staining;
step 7.1, collection of human sperm and neutrophils: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 7.2, sperm smear: uniformly dripping 0.5ml PBS solution containing 5×106 sperms on a polylysine coated glass slide, and naturally air-drying at room temperature to form a sperm smear;
step 7.3, wash 3 times with 0.01mph7.4PBS for 5 minutes each;
step 7.4, covering the mixture with 4% paraformaldehyde at room temperature for fixing for 15 minutes, and avoiding light;
step 7.5, washing 3 times by using 0.01MPH7.4PB PBS buffer solution for 5 minutes each time;
step 7.6, covering cells with 0.25% Triton X-100 (permeant) for 5-7 min (note: this step can be omitted without permeant);
step 7.7, 0.01MPH7.4PBS three washes of 5 minutes each;
step 7.8, goat sealing serum is sealed for 40 minutes at room temperature;
step 7.9, preparing a primary antibody, diluting SPAG8 to 200ug/ml, adding the primary antibody, and keeping out of light at 4 ℃ in a wet box for overnight;
step 7.10, washing with PBS three times, each for 5 minutes;
step 7.11, preparing fluorescence labeled secondary antibody (Ab 150079) coat anti-rabit IgG (H & L), diluting with PBS according to a ratio of 1:100, and preserving in a dark place; .
Step 7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (in the dark);
step 7.13, washing three times with PBS at 4 ℃ for 15 minutes (light shielding);
step 7.14, the DAPI is used for dying the nucleus, and the cells are completely covered (light shielding);
in step 7.15, the stained cells were observed with a confocal microscope, and cells exhibiting high-intensity red fluorescence (emission wavelength of 647 nm) were observed and photographed as an observation field.
Step 8, co-incubation experiment of human normal mature sperm and SPAG8 antibody
Step 8.1, collection of normal mature sperm and neutrophils of human origin: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 8.2, co-incubation of antibody and sperm: uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of SPAG8 antibody solution of 2mg/ml respectively, incubating at 37 ℃ for 30 minutes, washing with 0.01MPH7.4PBS, centrifuging for 3 times, and fixing volume to 10ml; the subsequent steps are the same as steps 7.2 to 7.15.
The invention has the advantages and beneficial effects that:
the anti-SPAG 8 antibody prepared by the invention not only has stable specific binding capacity with immobilized and denatured SPAG8 protein, but also can specifically bind with sperm membrane SPAG8 protein which keeps cell integrity in physiological state, thus obtaining good antibody orientation effect. The antibody can construct a composite targeting system by linking complex multicomponent and multifunctional biological groups at the Fc end according to specific application conditions so as to meet different requirements. The SPAG8 protein is widely distributed on the sperm membrane, so that the antibody prepared by the invention has good application prospect in the fields of targeted tracking, bio-pharmacy, accurate treatment and the like of sperms.
Drawings
FIG. 1 amino acid sequence of human SPAG8 and amino acid sequence of specific SPAG8 epitope.
FIG. 2TMHMM2.0 predicts that amino acids 1-485 of the SPAG8 protein are outside the cell membrane.
FIG. 3SIGnalP 4.1 shows that no signal peptide was found in the SPAG8 sequence.
FIG. 4ExPASY-ProtScale predicts that SPAG8 has 10 distinct hydrophobic regions, with amino acid at position 48 (Asp, aspartic acid) being the most hydrophobic. The 5 more distinct hydrophilic regions, the amino acid at position 406 (Gln, glutamine) is most hydrophilic.
FIG. 5ELSIA assay for SPAG8 antibody titers, (A) fourth week antibody titers of 1:800; (B) the eighth week antibody titer is 1:1600; (C) the ninth week antibody titer is 1:3200; (D) The titer at week ten was 1:3200, with the highest OD for the antibodies at week ten.
FIG. 6 as a negative control, anti-human IgG antibodies were not stained on human sperm.
In FIG. 7, SPAG8 protein is mainly distributed in the middle of the neck and tail of sperm, while partial protein is distributed in the tail of sperm, and neutrophils are not colored.
The immunofluorescence results of the anti-human sperm membrane SPAG8 antibody incubated with sperm in fig. 8 show that the epitope of the SPAG8 protein located outside the sperm cell membrane can bind directly to the antibody in the in vivo state of sperm.
Detailed Description
Example 1:
a preparation method of anti-human sperm membrane protein SPAG8 specific polypeptide and antibody specifically comprises the following steps:
step 1, analysis and design of specific antigen epitope of human sperm membrane protein SPAG8
Step 1.1, on-line prediction of transmembrane region of human sperm membrane protein SPAG8 Using TMHMM
Logging in a TMHMM main page, submitting a FASTA format SPAG8 protein sequence in a popup box of a 'select file' or pasting the sequence in a text box under 'OR by pasting sequence(s) in FASTA format'; out formats have three options, respectively, graphical output (existence); text output (no graphics); displaying proteins line by line (One line protein), and displaying a default option graphically; after the adjustment, the predicted analysis result is submitted and checked by pressing a submit bond, and the 1-485 positions of the SPAG8 protein sequence are all outside the cell membrane. (FIG. 2).
Step 1.2 predictive analysis of Signal peptide for human sperm Membrane protein SPAG8 Using SignalP 4.1Server
Logging in a SignalP 4.1Server main page, submitting a FASTA format protein sequence in a pop-up box of a 'selected file' or pasting the sequence in a text box below; parameter setting organosm group: selecting Eukaryotes; d-cutoff values: selecting Default (optimized for correlation); graphics output: selecting PNG (inline); output format: a Standard; method selection Input sequences may include TM regions. After the parameter setting is finished, submitting and checking a prediction analysis result by pressing a submit key, wherein the SPAG8 protein sequence does not find a signal peptide. (FIG. 3).
Step 1.3 predictive analysis of hydrophobicity of human sperm Membrane protein SPAG8 Using ExpASY-ProtScale
Logging in an ExPASY-ProtScale main interface, and inputting a protein sequence ID number in a text box of 'Enter a UniProtKB/Swiss-Prot or UniProtKB/TrEMBL accession number (AC)'; or the FASTA format protein sequence is stuck to a text box of Or you can paste your own sequence in the box below; the default setting is selected according to the parameter setting, the predicted analysis result is submitted and checked according to the 'sub', 10 obvious hydrophobic regions exist in SPAG8, and the amino acid (Asp, aspartic acid) at the 48 th position has the strongest hydrophobicity. The 5 more distinct hydrophilic regions, the amino acid at position 406 (Gln, glutamine) is most hydrophilic. (FIG. 4).
Logging in NCBI and UniProtKB protein databases, respectively taking SPAG8 and spim-associated antigen as keywords, selecting homosapiens from the species, inquiring the result through the protein database, and downloading a series of files in FASTA format of the membrane protein for subsequent analysis. The amino acid sequence of human sperm membrane protein SPAG8 is subjected to antigen epitope analysis by using biological information software, indexes such as transmembrane fragment, polypeptide activity, hydrophilicity, antigenicity and the like are evaluated, and the actual experience of preparing antibodies in the past is combined, so that the peptide segment of amino acid from 422 th to 437 th of the amino acid sequence of the human sperm membrane protein SPAG8 antigen epitope is finally determined, and the amino acid sequence of the synthesized polypeptide is determined to be GVSNIRTLDTPFRKNC (figure 1).
Step 2, synthesizing human SPAG8 polypeptide antigen and coupling purifying with KLH
Step 2.1 Synthesis of humanized SPAG8 Artificial polypeptide
10mg of purified SPAG8 (16 peptide) is synthesized by adopting an automatic polypeptide synthesizer, and purity identification is carried out by adopting mass spectrometry and high performance liquid chromatography, wherein the purity is more than 95%.
Step 2.2 Synthesis of polypeptide-KLH by EDC coupling
KLH was prepared as a 10mg/ml solution, SPAG8 (16 peptide) synthetic polypeptide was dissolved in 2.5ml of Imject@EDC Conjugation Bufjfer at a concentration of 0.4mg/ml, the polypeptide solution was added dropwise to the carrier protein solution, 10mg/ml EDC solution was prepared with ultrapure water, 250ul of this solution was immediately added to the mcKLH peptide solution, and after 2 hours of reaction at room temperature, the residue of EDC was removed.
Step 2.3, purification of the synthesized polypeptide-KLH Complex
60ml of ultra-pure water degassed by ultrasonic vibration is added into a Purification Buffer Salts bottle to dissolve the content, a desalting column is used for filtering every 0.5ml of sample, 0.5ml of synthesized polypeptide-KLH complex is gently dripped into 25ml of Purification Buffer Salts eluting column, standing is carried out for 2 minutes, 5ml of purification buffer is taken for eluting the column, the absorbance of the eluent is detected at 280nm by ultraviolet-visible absorption spectrometry, and the content of the polypeptide-KLH complex is calculated according to the absorption peak. If the immunogen needs to be stored for a longer period of time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing the artificial immunogen immune animal
Step 3.1, purchase 4 New Zealand white rabbits with SPF grade and weight of 2.0 KG. The normal serum was prepared by drawing 0.5ml from the auricular vein of animals before the immunization experiment, divided into SPAG8 group and control group;
step 3.2, antigen is encapsulated with adjuvant: before each immunization, 400-600ug of the SPAG8 polypeptide-KLH compound is inhaled into a 2.5ml sterile syringe, the other 2.5ml syringe is used for inhaling the equivalent amount of complete Freund adjuvant or Freund's incomplete adjuvant, the two syringes are connected by a sterile plastic hose and repeatedly pumped until the mixture is completely emulsified (one drop is not diffused in cold water any more, namely, the complete emulsification degree of water-in-oil is achieved), and the mixture can be used for immunization.
Step 3.3, animal sensitization: 400mg of polypeptide-KLH complex and complete Freund's adjuvant in a volume ratio of 1: step 2, mixing, namely performing animal sensitization after fully mixing, and injecting the mixture into the skin by adopting a multipoint method (about 200ul per point) on the trunk, neck and back of the animal in order to increase the sensitization effect;
step 3.4, cross-emulsifying antigen with Freund's incomplete adjuvant and Freund's complete adjuvant at the same antigen dose and boosting once a week, emulsification method was as before. The injection is respectively carried out at the subcutaneous parts of the back, the subcutaneous parts of the abdomen, the inguinal canal, the popliteal canal, the sole and other parts of the rabbit. During injection, the rabbit is fixed by the rabbit box, the skin is lifted by the left hand, the syringe is held by the right hand, the angle between the needle head and the skin is adjusted to be about 15 degrees, the needle head is injected into the skin for 1-2cm and then lifted upwards to prevent the needle head from penetrating into the muscle, and about 200ul of the needle head is injected at each point. 5-10ml of blood is taken from the rabbit ear vein after each immunization and is collected as immune serum;
step 3.5, specific immunization protocol is shown below, for a total of 10 weeks. Each rabbit was boosted twice by the ear vein at weeks 7 and 9. The Elsia experiments were performed at weeks 4,8, 9 and 10 to detect antibody titers and specificity, respectively.
Step 4, collecting rabbit anti-SPAG 8 polyclonal antibody serum
And 4.1, fixing four limbs of the rabbit in a supine position by using ropes, wherein the two upper limbs are fixed at the back of the head in a crossed manner, tying the upper jaw and the incisors of the rabbit by using the ropes, and pulling the rabbit backwards to fix the rabbit head on the two upper limbs in a homeopathic manner.
Step 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck along the middle of the neck from the upper fossa sternum to the lower jaw for about 15cm, carefully separating subcutaneous tissues along the trachea after finding out the trachea, and obtaining the mastoid muscle from the distal end to the throat and from the proximal end to the sternoclavicular muscle.
Step 4.3, the beating carotid artery is available below the trachea, the bilateral carotid arteries are carefully isolated and sufficiently dissociated.
Step 4.4, sleeving two black wires into one artery and separating the two black wires (one at the distal end and one at the proximal end), ligating the distal end of the artery by using the wires, clamping the proximal end of the artery by using an arterial clamp, cutting a small opening on the artery wall between the wires and the arterial clamp by using ophthalmic scissors, rapidly inserting a prefabricated thin plastic hose, and rapidly fixing the hose and the artery by using the proximal wire so as to prevent the hose from falling out and leaking blood.
Step 4.5, lightly loosening the arterial clamp, obliquely placing the arterial clamp by using a 50ml centrifuge tube, and increasing the blood discharge amount by treating the carotid artery at the other side in the same way until no blood drips out, and extruding the heart when the blood discharge amount is slow so as to increase the blood discharge amount. This method gave 75ml of SPAG8 rabbit blood.
Step 4.6, isolation and preservation of rabbit antisera rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first aspiration of serum, the serum was aspirated a second time by centrifugation at 12000 rpm at 4 degrees celsius for 15 minutes. Finally, about 50ml of SPAG8 antibody serum is obtained, and after being split-packed by a 15ml centrifuge tube, naN3 with the volume of 0.01 percent is added respectively, and the mixture is placed into a refrigerator with the temperature of minus 20 ℃ for storage.
Step 5, separating and purifying antibody by saturated ammonium sulfate method
Step 5.1, 3ml of rabbit antiserum was transferred into a 15ml centrifuge tube, 3ml of 0.01M pH7.4PBS was gradually added at 4℃and 6ml of saturated ammonium sulfate solution at pH7.0 was gradually added dropwise with slow shaking.
Step 5.2 when the ammonium sulphate solution reached 50% saturation, the solution was left at 4℃overnight.
Step 5.3, centrifuging the solution at 4 ℃ for 20min at 10000 revolutions per minute, and removing the supernatant to obtain the globulin precipitate.
Step 5.4, after dissolving the precipitate in 3ml of 0.01M PH7.4PBS at 4 ℃, 1.5ml of saturated ammonium sulfate solution (33% saturation of the ammonium sulfate solution) was added dropwise, and the precipitate was taken for 30min. The procedure was repeated twice.
Step 5.5, centrifuging at 4 ℃ for 10min at 10000 revolutions per minute, and discarding the supernatant to obtain antibody IgG precipitate.
Step 5.6, the supernatant was discarded, 3ml of 0.01M PH7.4PBS was added to dissolve the antibody IgG pellet, and the pellet was filled into dialysis bags.
Step 5.7, dialyzing the dialysis bag in 0.01M PH7.4PBS solution at 4deg.C. The liquid is changed for several times until the external liquid for dialysis has no yellow change. 8) And taking a sample in the dialysis bag, drying in vacuum, taking a small amount of sample, diluting by a proper multiple, and measuring the protein content.
Step 6, enzyme-linked immunosorbent assay identification of serum SPAG8 antibody and natural human SPAG8 protein binding titer step 6.1, preparation of human sperm membrane protein soluble antigen
After 3-5d of forbidden appetite of healthy men, the masturbation method takes semen. After the sperm is liquefied in a water bath at 37 ℃, carrying out routine semen analysis, and collecting semen with the sperm survival rate of more than 60 percent, the sperm motility a grade of more than 25 percent, or the sperm with the sperm motility a grade of more than 50 percent for standby; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form two layers of Percoll density gradient separation columns; 4ml of semen to be used was carefully added to the centrifuge tube and a clear interface was seen. 3000 rpm, centrifuging for 15min, carefully sucking out sperm layer, adding into 2ml PBS, centrifuging at 4deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Clean sperm cell pellet was obtained and thoroughly mixed with about 3-5ml PBS and transferred to a 15ml sterile centrifuge tube to obtain sperm cell suspension.
Step 6.1.2, breaking up sperm cell membranes by using an ultrasonic cytoclasis instrument, and fully exposing antigenic sites by membrane proteins. The method comprises the following steps: 3-5ml of a 15ml centrifuge tube containing sperm cells was placed in crushed ice, the top cap was opened, an ultrasonic cytobreaker probe was placed in the solution, the intensity was adjusted to 70%, and the breaking time was 6min (breaking 5s, suspending 5s to prevent temperature superantigen denaturation and foam generation).
Step 6.1.3, centrifuging the solution (4 ℃ C., 12000 r/min), discarding the precipitate, collecting supernatant (sperm-soluble membrane protein antigen), and preserving at 4 ℃ C. Repeating the steps, collecting the soluble antigens obtained in the steps and storing the soluble antigens in a refrigerator at the temperature of-80 ℃.
Step 6.2, coating, namely diluting the humanized sperm membrane protein antigen to 50ug/ml, dripping the diluted humanized sperm membrane protein antigen into a plurality of small holes of a 96-well plate, 100 mu L/hole and standing at 4 ℃ overnight.
Step 6.3, sealing: the soluble antigen in the wells was discarded, and 150. Mu.L/well of 5% nonfat dry milk (blocking solution) was added and incubated at 37℃for 40min.
Step 6.4, washing, namely adding 200 ul/hole of washing liquid (PBST), washing three times, and standing for 3min each time.
Step 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of serum antibodies to be tested with different dilutions, and standing at 37 ℃ for 40min.
And 6.6, washing, namely adding PBST into the small holes, standing for 3min, discarding the washing liquid in the holes, repeating for three times, and finally fully throwing away the washing liquid in the holes.
Step 6.7, adding enzyme-labeled antibody (secondary antibody) by diluting HRP-labeled goat anti-rabbit secondary antibody by 1:10000, adding 100 μl/well, and standing at 37deg.C for 40min.
Step 6.8, wash with PBST three times, 3min each time.
And 6.9, developing, namely adding 100 mu L/hole of TMB developing solution, and standing for 15min at normal temperature in a dark place.
Step 6.10, stop solution, 1M H2SO4, 50. Mu.L/well.
Step 6.11, measuring, namely measuring the OD 490nm value by using an enzyme-labeled instrument. After ten times of immunization, the titers of the SPAG8 antibody at the fourth week, the eighth week, the ninth week and the tenth week are respectively 1:800, 1:1600, 1:3200 and 1:3200, and the OD value of the antibody at the tenth week is highest; (FIG. 5).
Step 7, positioning SPAG8 protein distribution on sperm surface by cell immunofluorescence staining
Step 7.1, collection of human sperm and neutrophils: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times;human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 7.2, sperm smear: uniformly dripping 0.5ml PBS solution containing 5×106 sperms on a polylysine coated glass slide, and naturally air-drying at room temperature to form a sperm smear;
step 7.3, wash 3 times with 0.01mph7.4PBS for 5 minutes each;
step 7.4, covering the mixture with 4% paraformaldehyde at room temperature for fixing for 15 minutes, and avoiding light;
step 7.5, washing 3 times by using 0.01MPH7.4PB PBS buffer solution for 5 minutes each time;
step 7.6, covering cells with 0.25% Triton X-100 (permeant) for 5-7 min (note: this step can be omitted without permeant);
step 7.7, 0.01MPH7.4PBS three washes of 5 minutes each;
step 7.8, goat sealing serum is sealed for 40 minutes at room temperature;
step 7.9, preparing a primary antibody, diluting SPAG8 to 200ug/ml, and keeping the wet box at 4 ℃ overnight in a dark place;
step 7.10, washing with PBS three times, each for 5 minutes;
step 7.11, preparing fluorescence labeled secondary antibody (Ab 150079) coat anti-rabit IgG (H & L), diluting with PBS according to a ratio of 1:100, and preserving in a dark place; .
Step 7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (in the dark);
step 7.13, washing three times with PBS at 4 ℃ for 15 minutes (light shielding);
step 7.14, the DAPI is used for dying the nucleus, and the cells are completely covered (light shielding);
in step 7.15, the stained cells were observed with a confocal microscope, and cells exhibiting high-intensity red fluorescence (emission wavelength of 647 nm) were observed and photographed as an observation field. Immunofluorescent staining of human sperm and neutrophil smears showed that IgG antibodies were not stained on human sperm (fig. 6); SPAG8 protein was mainly distributed in the middle of the neck and tail of sperm, with some protein distributed in the tail of sperm, neutrophils were not stained (fig. 7);
step 8, co-incubation experiment of human normal mature sperm and SPAG8 antibody
Step 8.1, collection of normal mature sperm and neutrophils of human origin: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 8.2, co-incubation of antibody and sperm: uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of SPAG8 antibody solution of 2mg/ml respectively, incubating at 37 ℃ for 30 minutes, washing with 0.01MPH7.4PBS, centrifuging for 3 times, and fixing volume to 10ml; the subsequent operation steps are the same as the 7.2 th to 7.15 th steps; the co-incubation experiment of the SPAG8 antibody and the sperm can observe the expression condition of the antibody combined with the motile sperm SPAG8 protein, and the co-incubation experiment shows that the antibody generates fluorescence at the corresponding part of the sperm, which indicates that the epitope is positioned outside the sperm cell membrane and can be directly combined with the antibody under the sperm living state (figure 8).
Claims (7)
1. The human sperm membrane protein SPAG8 specific polypeptide is characterized in that antigen binding sites are widely distributed on the neck, the middle section and the outer membrane of the tail of human sperm, and the amino acid sequence of the polypeptide is as follows: GVSNIRTLDTPFRKNC.
2. A method for preparing an antibody against human sperm membrane protein SPAG8, which is characterized by comprising the following steps:
1) Analysis and design of human sperm membrane protein SPAG8 specific polypeptides of claim 1;
2) Synthesizing human sperm membrane protein SPAG8 specific polypeptide antigen and coupling and purifying carrier protein;
3) Preparing an artificial immunogen immune animal;
4) Collecting antibody serum;
5) Separating and purifying an anti-human sperm membrane protein SPAG8IgG antibody by a saturated ammonium sulfate method;
6) The ELISA experiments are used for identifying the binding titer of the serum antibody and the natural human SPAG8 protein;
7) Cell immunofluorescence staining locates SPAG8 protein distribution on sperm surface.
3. The method of claim 2, wherein the carrier protein is Keyhole Limpet Hemocyanin (KLH).
4. The method of claim 2, wherein the artificial immunogen is KLH-polypeptide mixed with complete or incomplete freund's adjuvant to immunize the animal.
5. The method for preparing the antibody according to claim 2, wherein the carrier protein is coupled and purified to synthesize the antigen polypeptide, the amino acid hydrophobe of the polypeptide is covalently crosslinked with the amino group of the carrier protein through a crosslinking agent, and the antigen polypeptide is purified through a chromatographic column.
6. The method of claim 2, wherein the immunized animal is a human sperm membrane protein extract having a serum titer greater than 1 after multiple subcutaneous injections in the back of the animal and two or more booster immunizations: 3200.
7. the method for producing an antibody according to claim 2, wherein the isolated and purified antibody is an IgG antibody which can be obtained in high purity from the serum of the antibody by ammonium persulfate precipitation and protein affinity purification.
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| CN1667121A (en) * | 2004-03-11 | 2005-09-14 | 中国医学科学院基础医学研究所 | Biology function of encoded protein of HSD-1(SPAG8) gene specific for human testis |
| CN1869065A (en) * | 2005-05-24 | 2006-11-29 | 中国医学科学院基础医学研究所 | Human testis specific expression protein SPAG8 for taking part in regulating and controlling sperm generation |
| CN108148127A (en) * | 2016-12-05 | 2018-06-12 | 天津奥维亚生物技术有限公司 | A kind of people MAP3K7IP2 polypeptides and its preparation method for antibody |
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| CN1667121A (en) * | 2004-03-11 | 2005-09-14 | 中国医学科学院基础医学研究所 | Biology function of encoded protein of HSD-1(SPAG8) gene specific for human testis |
| CN1869065A (en) * | 2005-05-24 | 2006-11-29 | 中国医学科学院基础医学研究所 | Human testis specific expression protein SPAG8 for taking part in regulating and controlling sperm generation |
| CN108148127A (en) * | 2016-12-05 | 2018-06-12 | 天津奥维亚生物技术有限公司 | A kind of people MAP3K7IP2 polypeptides and its preparation method for antibody |
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