CN113896769A - 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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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- 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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- C07—ORGANIC CHEMISTRY
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- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
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Abstract
A method for preparing human sperm membrane protein SPAG8 specific polypeptide and antibody is provided. The C-terminal specific polypeptide amino acid sequence is as follows: GVSNIRTLDTPFRKNC are provided. The preparation of the anti-human sperm membrane protein SPAG8 antibody comprises the following steps: (1) analyzing and designing a specific extracellular epitope; (2) synthesizing specific artificial polypeptide; (3) crosslinking the synthetic polypeptide with carrier protein; (4) preparing rabbit polyclonal antibody; (5) collecting, separating and purifying to obtain the antibody. The specific antihuman sperm membrane protein SPAG8 antibody prepared by the invention has high titer, strong affinity and good specificity, can be specifically combined with the epitope of the human sperm membrane protein SPAG8 with natural activity in vivo and in vitro, and can be used for fluorescence 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 protein, but also has application prospects in the fields of targeted tracking, biopharmaceuticals, precise 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, and has good application prospects in the fields of targeted tracing, biological pharmacy, precise treatment and the like.
Background
As the research of specific antigen sites and antibodies of sperm membrane proteins is lacked, the mechanism of action of the specific antigen sites of sperm apical area, neck and tail sperm surface membrane proteins in the process of sexual reproduction is not clear, a series of scientific problems such as construction of specific immune infertility targeted treatment method and the like are further disclosed by means of interdisciplinary application of technologies such as molecular biology, bioinformatics, high-throughput sequencing and the like, and the method is a series of scientific problems of promotion of healthy fertility, and the like, And constructing a great demand for population safety.
(1) Function of SPAG 8: the human sperm membrane protein SPAG8 (SPAG 8) gene is located in the antisense strand of 9p13.3 DNA, is 4,488bp in length and has 9 exons, and two mRNA subtypes have been found: NM _001039592.1 and NM _ 172312.1. The SPAG8 protein product has 426 amino acids, molecular weight of 44,819Da, and is divided into 2 subtypes, which are single transmembrane II-type membrane protein and secretory protein. The SPAG8 protein is widely distributed in cytoplasm, nucleus, vesicles, and acrosome, and also in cytoskeleton, microtubule tissue center, mitotic spindle. In mature sperm cells, distribution was detected in the acrosomal region of the head and the mid-portion of the tail.
(2) Information on the SPAG8 antibody product: through search, the SPAG8 protein polyclonal antibody is on the market, but the epitope of the antibody is different and can only be detected in vitro.
(3) SPAG8 and diseases: 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. The mitotic cycle, SPAG8, is localized at the microtubule tissue center (MTOC). The middle stage extends to the spindle microtube; later on detectable in the star microtubules and the intermediate zone; the end stage remains in the middle zone. After cell division, and back to the MTOC, the protein encoded by the gene is localized on the sperm surface. In spermatogenesis, SPAG8 plays a role in regulating tau transcriptional activation by enhancing co-activator FHL5 binding to cyclic adenosine monophosphate response element regulator subtype tau and enhancing FHL5 regulation of tau transcriptional activation, in addition, SPAG8 is involved in acrosome reactions and binding of sperm to zona pellucida, plays a role in regulating progression of G2/M phase by delaying activation of CDK1 required for mitosis into the mitotic phase, and SPAG8 may also influence fertility by interacting with RANBP9, which plays a role in microtubule formation.
Disclosure of Invention
The invention aims at overcoming the defects of an AsAb preparation method for immune sterility mechanism research and provides a preparation method of a human seminal plasma membrane protein SPAG8 polypeptide and an antibody. Different from in vitro experiment detection antibodies, the anti-SPAG 8 antibody prepared by the polypeptide can specifically recognize natural human SPAG8 protein in tissues or cells through immunofluorescence and enzyme-linked immunosorbent assay on one hand, can perform research on an anti-sperm antibody mediated immune sterility action mechanism by means of specific surface antigen sites of SPAG8 on the other hand, and can also perform research on the aspects of targeted tracing, biopharmaceutical and precise treatment and the like.
Technical scheme of the invention
The specific polypeptide of human sperm membrane protein SPAG8 has antigen combining sites widely distributed in the neck, middle tail and outer tail membranes of human sperm, and the polypeptide sequence is the peptide segment from 422 th to 437 th amino acids in the amino acid sequence of human sperm membrane protein SPAG8, and the amino acid sequence of the polypeptide is: GVSNIRTLDTPFRKNC are provided.
The invention also provides a preparation method of the antibody of the anti-human sperm membrane protein SPAG8, which comprises the following steps:
the amino acid sequence of the protein is subjected to epitope analysis by using bioinformatics software, and transmembrane segments, polypeptide activity, hydrophilicity, antigenicity and other indexes 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 'selection file' pop-up box OR pasting the sequence in an 'OR by mapping sequence(s) in FASTA format:' a lower text box; out format has three options, respectively graphics output (Extensive, with graphics); text output (extend, no graphics); displaying the protein line by line (One line protein), and displaying the default option graphically; and after the adjustment is finished, a submit key is pressed to submit and check the prediction analysis result.
Step 1.2, predictive analysis of the Signal peptide of Membrane proteins Using SignalP 4.1Server
Logging in a SignalP 4.1Server main page, and submitting a FASTA format protein sequence in a 'selection file' pop-up box or pasting the sequence in a text box below; parameter setting organization group: selecting euryotes; d-cutoff values: selecting default (optimized for correlation); graphics output: selecting PNG (inline); output format: standard; the Method selects Input sequences may include TM regions. And after the parameter setting is finished, submitting the parameters by pressing a submit key and checking a prediction analysis result.
Step 1.3, predictive analysis of the 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 access number (AC)'; or pasting the protein sequence in the FASTA format in a text box of 'Or you can past you sequence in the box below'; and selecting default settings for parameter setting, and submitting and checking the prediction analysis result according to the Submit after the setting is finished.
step 2.1, Synthesis of SPAG8 Artificial polypeptide
And (3) synthesizing and purifying by adopting an automatic polypeptide synthesizer, and identifying the purity by adopting mass spectrometry and high performance liquid chromatography, wherein the purity is more than 95%.
Step 2.2, polypeptide-KLH is synthesized by EDC coupling
KLH was prepared as a 10mg/ml solution, the synthesized 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, a 10mg/ml EDC solution was prepared with ultrapure water, 250. mu.l of this solution was immediately added to the mcKLH peptide solution, and after 2 hours at room temperature, the synthesized polypeptide-KLH complex was obtained and the residue of EDC was removed.
Step 2.3, purification and Synthesis of polypeptide-KLH Complex
Adding 60ml of ultrapure water subjected to ultrasonic oscillation degassing into a Purification Buffer Salts bottle to dissolve the content, filtering each 0.5ml of sample by using a desalting column, slightly dropwise adding 0.5ml of the synthesized polypeptide-KLH compound into 25ml of Purification Buffer Salts elution column, standing for 2 minutes, taking 5ml of Purification Buffer solution to elute the column, collecting the eluate, measuring absorbance at 280nm by using an ultraviolet visible absorption spectrum, and calculating the content of the polypeptide-KLH compound according to an absorption peak. If the immunogen needs to be stored for a longer time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing artificial immunogen immune animals;
at step 3.1, 4 new Zealand white rabbits of SPF grade with a weight of 2.0KG were purchased. Dividing into experimental group and control group, and preparing normal serum from animal ear edge vein blood 0.5ml before immunity experiment;
3.2, wrapping the antigen by using an adjuvant: before each immunization, 600ug of the polypeptide-KLH complex is sucked into a 2.5ml sterile syringe, the other 2.5ml syringe is used for sucking the same amount of complete Freund's adjuvant or Freund's incomplete adjuvant, the two syringes are connected by a sterile plastic hose and are repeatedly pumped until complete emulsification (the condition that one drop of the polypeptide-KLH complex is not diffused in cold water is reached, namely the complete emulsification degree of water-in-oil) is achieved, and then the polypeptide-KLH complex can be used for immunization.
3.3, animal sensitization: 400mg of polypeptide-KLH complex and complete Freund's adjuvant in a volume ratio of 1:1, mixing, performing the operation of step 2, fully mixing, performing animal sensitization, and injecting the mixture into the body, neck and back of an animal subcutaneously by a multipoint method (each point is about 200ul) in order to increase the sensitization effect;
step 3.4, antigen was boosted weekly at the same antigen dose and with Freund's incomplete adjuvant and Freund's complete adjuvant, the emulsification procedure being as before. Respectively injecting into the rabbit at subcutaneous back, subcutaneous abdomen, groin, popliteal groove, and sole. During injection, a rabbit is fixed by a rabbit box, the skin is lifted by the left hand, the injector 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 is lifted upwards to prevent the needle head from penetrating into muscles, and about 200ul of injection is injected at each point.
Step 3.5, the specific immunization protocol, is shown below, for a total of 10 weeks. Each rabbit was boosted twice at weeks 7 and 9 with the marginal veins of the ear. The Elsia experiments were performed at weeks 4,8, 9 and 10 to detect antibody titer and specificity, respectively.
Step 4, collecting antibody serum;
and 4.1, fixing four limbs of the rabbit in the supine position by using ropes, wherein the two upper limbs are crossed and arranged behind the head to be fixed, tying the upper jaw incisors of the rabbit by using a string and pulling the upper jaw incisors backwards, and fixing the rabbit head on the two upper limbs in a homeopathic manner.
And 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck by about 15cm from the suprasternal fossa to the lower jaw along the center of the neck, carefully separating subcutaneous tissues along the trachea after finding the trachea, and leading the far end of the trachea to the throat and the near end of the trachea to the sternocleidomastoid muscle.
And 4.3, showing a pulsating stiff artery below the trachea, carefully separating the carotid arteries on both sides, and fully dissociating.
And 4.4, sleeving two black silk threads into one side artery, separating the two black silk threads (one at the far end and the other at the near end), ligating the far end of the artery by using a silk thread, clamping the near end of the artery by using an artery clamp, shearing a small opening on the artery wall between the silk thread and the artery clamp by using an ophthalmic scissors, quickly inserting a prefabricated thin plastic hose, and quickly fixing the hose and the artery by using the near end silk thread to prevent the hose from dropping out and blood leakage.
And 4.5, slightly loosening the artery clamp, obliquely placing a 50ml centrifuge tube to receive arterial blood ejected from the blood-letting tube until no blood drips out, treating the carotid artery on the other side by the same method to increase the blood-letting amount, and pressing the heart when the blood-letting flow is slow to increase the blood-letting amount.
Step 4.6 isolation and storage of Rabbit antiserum Rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first serum aspiration, the serum was centrifuged at 12000 rpm at 4 ℃ for 15min, and the serum aspirated for the second time. And finally obtaining about 50ml of SPAG8 antibody serum, subpackaging by using a 15ml centrifuge tube, respectively adding 0.01% NaN3 by volume, and storing in a refrigerator at the temperature of-20 ℃.
5, separating and purifying the anti-human sperm membrane protein SPAG8IgG antibody by a saturated ammonium sulfate method;
at step 5.1, 3ml of rabbit antiserum was transferred to a 15ml centrifuge tube, 3ml of 0.01M pH7.4PBS was gradually added at 4 ℃ and 6ml of a saturated ammonium sulfate solution at pH7.0 was added dropwise with gentle shaking.
At step 5.2, when the ammonium sulfate solution reached 50% saturation, the solution was left at 4 ℃ overnight.
And 5.3, placing the solution at 4 ℃, centrifuging for 20min at 10000 r/min, and removing supernatant to obtain globulin precipitate.
After dissolving the precipitate in 3ml of 0.01M pH7.4PBS at 4 ℃ in step 5.4, 1.5ml of a saturated ammonium sulfate solution was added dropwise (to bring the ammonium sulfate solution to 33% saturation), and the precipitate was allowed to settle for 30 min. This was repeated twice.
Centrifuging at 10000 rpm for 10min at 4 ℃ in the step 5.5, and removing the supernatant to obtain the antibody IgG precipitate.
At step 5.6, the supernatant was discarded, and 3ml of 0.01M pH7.4PBS was added to dissolve the antibody IgG precipitate, and the mixture was packed into a dialysis bag.
At step 5.7, the dialysis bag was dialyzed at 4 ℃ against 0.01M PBS solution (pH 7.4). During the period, the solution was changed several times until the external dialysate had no yellow change. 8) And (3) taking a sample in the dialysis bag, drying in vacuum, taking a small amount of the sample, diluting by proper times, and then determining the protein content.
Step 6, identifying the binding titer of the serum antibody and the natural human SPAG8 protein by an enzyme-linked immunosorbent assay;
step 6.1, preparation of human sperm membrane protein soluble antigen
After abstinence for 3-5 days in step 6.1.1, semen is collected by masturbation. After the sperms are 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 of a grade of more than 25 percent, or the a + b grade of more than 50 percent for later use; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form a two-layer Percoll density gradient separation column; 4ml of the ready-to-use semen were carefully added to the centrifuge tube, and a clear interface was visible. Centrifuging at 3000 rpm for 15min, carefully sucking sperm layer, adding into 2ml PBS solution, centrifuging at 4 deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4 deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Obtaining a clean sperm cell precipitate, fully mixing the precipitate with about 3-5ml PBS, and transferring the mixture into a 15ml sterile centrifuge tube to obtain a sperm cell suspension.
And 6.1.2, breaking the cell membrane of the sperm by using an ultrasonic cell disruptor, and fully exposing the antigen sites by using membrane protein. The method comprises the following steps: a15 ml centrifuge tube containing sperm cells (3-5 ml) was placed in crushed ice with the top cap open and the ultrasonic cell disruptor probe placed in solution with intensity adjusted to 70% for 6min (disruption for 5s, pause for 5s to prevent over-temperature antigen denaturation and foam generation).
And 6.1.3, centrifuging the solution (4 ℃, 12000 r/min), discarding the precipitate, taking the supernatant (sperm soluble membrane protein antigen), and storing at 4 ℃. Repeating the above steps, collecting the soluble antigens obtained in different times, and storing in a-80 deg.C refrigerator.
6.2, coating, namely, diluting the human sperm membrane protein antigen to 50ug/ml, dripping the diluted human sperm membrane protein antigen into a plurality of small holes of a 96-well plate, and keeping the diluted human sperm membrane protein antigen at 100 mu L/hole overnight at 4 ℃.
And 6.3, sealing: the soluble antigen in the wells was discarded, 150. mu.L/well of 5% skim milk powder (blocking solution) was added, and incubation was carried out at 37 ℃ for 40 min.
And 6.4, washing, namely adding 200ul of washing solution (PBST) per hole, washing for three times, and standing for 3min each time.
And 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of the serum antibody to be detected with different dilutions, and standing at 37 ℃ for 40 min.
And 6.6, washing, namely adding PBST into the small holes, keeping the small holes at 200 ul/hole, standing for 3min, discarding the washing liquid in the holes, repeating the steps for three times, and finally fully throwing the washing liquid in the holes.
Step 6.7, enzyme-labeled antibody (secondary antibody) is added, and HRP-labeled goat anti-rabbit secondary antibody is diluted at a ratio of 1:10000, added to 100. mu.L/well, and left at 37 ℃ for 40 min.
And 6.8, washing, namely washing three times by PBST for 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, adding 1M H2SO4, 50 μ L/well.
And 6.11, measuring by using an enzyme-linked immunosorbent assay (ELISA) instrument to determine the OD 490nm value.
7, positioning the SPAG8 protein distribution on the surface of the sperm by cell immunofluorescence staining;
7.1, collecting human sperms and neutrophils: collecting semen of healthy people by masturbation, completely liquefying at 37 deg.C, centrifuging at 2000 rpm for 10min to collect spermatid, 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 0.01MPH7.4 PBS. Counting with a blood counting chamber, and pulverizing sperm and neutrophilic granulomaCell concentration was adjusted to 2X 106Per ml;
step 7.2, sperm smear: uniformly dripping 0.5ml of PBS solution containing 5 x 106 sperms on a polylysine coated glass slide, and naturally drying at room temperature to form a sperm smear;
7.3, washing with 0.01MPH7.4PBS for 3 times, each time for 5 minutes;
7.4, covering the mixture with 4 percent paraformaldehyde for fixing for 15 minutes at room temperature, and keeping the mixture out of the sun;
7.5, washing with 0.01MPH7.4PB PBS buffer solution for 3 times, each time for 5 minutes;
step 7.6, 0.25% Triton X-100 (permeant) covering the cells for 5-7 minutes (note: this step can be omitted without adding permeant);
7.7, washing for three times with 0.01MPH7.4PBS for 5 minutes each time;
7.8, sealing the goat serum at room temperature for 40 minutes;
7.9, preparing a primary antibody, diluting the SPAG8 to 200ug/ml, and adding the mixture in a wet box at 4 ℃ in a dark place overnight;
7.10, washing with PBS for three times, 5 minutes each time;
7.11, preparing a fluorescence labeled secondary antibody (Ab150079) Goat anti-rabbitIgG (H & L), diluting the mixture with PBS according to a ratio of 1:100, and storing the mixture in a dark place; .
7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (keeping out of light);
7.13, washing three times with 4 ℃ PBS, each time for 15 minutes (protected from light);
step 7.14, staining the nuclei with DAPI, and completely covering the cells (keeping out of the sun);
at step 7.15, the staining result was observed by using a confocal microscope, and cells showing high-intensity red fluorescence (emission wavelength: 647nm) were observed as a visual field, and photographed.
Step 8.1, collecting human normal mature sperms and neutrophils: collecting semen of healthy human by masturbation, completely liquefying at 37 deg.C, centrifuging at 2000 rpm for 10min to collect sperm cells, washing with 0.01MPH7.4PBS, and centrifuging3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01MPH7.4 PBS. Counting with a blood counting chamber, adjusting the concentration of sperm and neutrophil to 2 × 106Per ml;
step 8.2, antibody and sperm co-incubation: respectively and uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of 2mg/ml SPAG8 antibody solution, incubating for 30 minutes at 37 ℃, washing and centrifuging for 3 times by using 0.01MPH7.4PBS, and fixing the volume to 10 ml; the subsequent operation steps are the same as the above 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 can have stable specific binding capacity with fixed denatured SPAG8 protein, but also can be specifically bound with sperm membrane SPAG8 protein which keeps the integrity of cells under physiological state, thereby obtaining good antibody orientation effect. The antibody can meet different requirements by constructing a composite targeting system through an Fc end linked with a complex multi-component and multifunctional biological group according to specific application conditions. The SPAG8 protein is widely distributed on a sperm membrane, so that the antibody prepared by the invention has good application prospects in the fields of sperm targeted tracking, biopharmaceuticals, precise treatment and the like.
Drawings
Figure 1 amino acid sequence of humanized 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.
Figure 3SignalP 4.1 shows that no signal peptide is found in the sequence of SPAG 8.
FIG. 4ExPASY-ProtScale predicts that there are 10 distinct hydrophobic regions of SPAG8, with the amino acid at position 48 (Asp, aspartic acid) being the most hydrophobic. The 5 more obvious hydrophilic regions, the amino acid at position 406 (Gln, glutamine) is most hydrophilic.
FIG. 5ELSIA determination of SPAG8 antibody titers, (A) the fourth week antibody titers were 1: 800; (B) the eighth week antibody titer was 1: 1600; (C) the ninth week antibody titer was 1: 3200; (D) the titers at week ten were 1:3200, respectively, and the antibody OD values were highest at week ten.
FIG. 6 shows that, as a negative control, no staining of the human-derived sperm was observed with the anti-human IgG antibody.
FIG. 7SPAG8 protein was mainly distributed in the neck and middle tail of sperm, and some protein was distributed in the tail of sperm, and no staining of neutrophils was observed.
FIG. 8 shows that the SPAG8 protein can be directly combined with antibody at the antigenic determinant outside the sperm cell membrane under the living state of the sperm as shown by the immunofluorescence result of the incubation of the SPAG8 antibody and the sperm against the human sperm membrane.
Detailed Description
Example 1:
a preparation method of anti-human sperm membrane protein SPAG8 specific polypeptide and antibody specifically comprises the following steps:
Step 1.1, using TMHMM to predict the transmembrane region of human sperm membrane protein SPAG8 on line
Logging in a TMHMM main page, submitting a FASTA format SPAG8 protein sequence in a 'selection file' pop-up box OR pasting the sequence in an 'OR by mapping sequence(s) in FASTA format:' a lower text box; out format has three options, respectively graphics output (Extensive, with graphics); text output (extend, no graphics); displaying the protein line by line (One line protein), and displaying the default option graphically; after the adjustment is finished, submitting the protein by pressing a submit key and checking a prediction analysis result, wherein 1-485 sites of the SPAG8 protein sequence are all positioned outside the cell membrane. (FIG. 2).
Step 1.2, using SignalP 4.1Server to carry out prediction analysis of signal peptide on human sperm membrane protein SPAG8
Logging in a SignalP 4.1Server main page, and submitting a FASTA format protein sequence in a 'selection file' pop-up box or pasting the sequence in a text box below; parameter setting organization group: selecting euryotes; d-cutoff values: selecting default (optimized for correlation); graphics output: selecting PNG (inline); output format: standard; the Method selects Input sequences may include TM regions. After the parameters are set, submitting the parameters by pressing a submit key and checking a prediction analysis result, wherein the SPAG8 protein sequence does not find a signal peptide. (FIG. 3).
Step 1.3, the hydrophobicity prediction analysis of human sperm membrane protein SPAG8 by 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 access number (AC)'; or pasting the protein sequence in the FASTA format in a text box of 'Or you can past you sequence in the box below'; the default setting is selected for parameter setting, after the setting is finished, the result of prediction analysis is submitted according to 'Submit', and 10 obvious hydrophobic regions exist in the SPAG8, and the amino acid (Asp and aspartic acid) at the 48 th position has the highest hydrophobicity. The 5 more obvious hydrophilic regions, the amino acid at position 406 (Gln, glutamine) is most hydrophilic. (FIG. 4).
Logging in NCBI and UniProtKB protein databases, selecting 'Homo sapiens' by using 'SPAG 8' and 'sphere-associated antigen 8' as key words, querying results through the protein databases, and downloading a series of files of the membrane protein in the FASTA format for subsequent analysis. The method comprises the steps of utilizing biological information software to carry out antigen epitope analysis on the amino acid sequence of the human sperm membrane protein SPAG8, evaluating transmembrane fragments, polypeptide activity, hydrophilicity, antigenicity and other indexes, combining practical experience of preparing antibodies in the past, finally determining the peptide segment of the human sperm membrane protein SPAG8 antigen epitope from the 422 th amino acid to the 437 th amino acid of the amino acid sequence, and determining the synthetic polypeptide amino acid sequence as GVSNIRTLDTPFRKNC (figure 1).
Step 2.1, Synthesis of human SPAG8 Artificial polypeptide
10mg of purified SPAG8(16 peptide) is synthesized by adopting an automatic polypeptide synthesizer, and the purity is identified by adopting mass spectrometry and high performance liquid chromatography, wherein the purity is more than 95%.
Step 2.2, polypeptide-KLH is synthesized 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, a 10mg/ml solution of EDC was prepared with ultrapure water, 250ul of this solution was immediately added to the mcKLH peptide solution, and the residue of EDC was removed after 2 hours reaction at room temperature.
Step 2.3, purification and Synthesis of polypeptide-KLH Complex
Adding 60ml of ultrapure water subjected to ultrasonic oscillation degassing into a Purification Buffer Salts bottle to dissolve the content, filtering each 0.5ml of sample by using a desalting column, slightly dropwise adding 0.5ml of the synthesized polypeptide-KLH compound into 25ml of Purification Buffer Salts elution column, standing for 2 minutes, taking 5ml of Purification Buffer solution to elute the column, collecting the eluate, measuring absorbance at 280nm by using an ultraviolet visible absorption spectrum, and calculating the content of the polypeptide-KLH compound according to an absorption peak. If the immunogen needs to be stored for a longer time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing artificial immunogen immune animal
At step 3.1, 4 new Zealand white rabbits of SPF grade with a weight of 2.0KG were purchased. Dividing into SPAG8 group and control group, and preparing normal serum from animal ear edge vein blood 0.5ml before immune experiment;
3.2, wrapping the antigen by using an adjuvant: before each immunization, 600ug of SPAG8 polypeptide-KLH complex is sucked into a 2.5ml sterile syringe, the other 2.5ml syringe is used for sucking the same amount of complete Freund's adjuvant or Freund's incomplete adjuvant, the two syringes are connected by a sterile plastic hose and are repeatedly pumped until complete emulsification (dripping one drop into cold water and no diffusion, namely reaching the complete emulsification degree of water-in-oil), and the mixture can be used for immunization.
3.3, animal sensitization: 400mg of polypeptide-KLH complex and complete Freund's adjuvant in a volume ratio of 1:1, mixing, performing the operation of step 2, fully mixing, performing animal sensitization, and injecting the mixture into the body, neck and back of an animal subcutaneously by a multipoint method (each point is about 200ul) in order to increase the sensitization effect;
step 3.4, antigen was boosted weekly at the same antigen dose and with Freund's incomplete adjuvant and Freund's complete adjuvant, the emulsification procedure being as before. Respectively injecting into the rabbit at subcutaneous back, subcutaneous abdomen, groin, popliteal groove, and sole. During injection, a rabbit is fixed by a rabbit box, the skin is lifted by the left hand, the injector 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 is lifted upwards to prevent the needle head from penetrating into muscles, and about 200ul of injection is injected at each point. Collecting 5-10ml of blood from marginal vein of rabbit after each immunization as immune serum;
step 3.5, the specific immunization protocol, is shown below, for a total of 10 weeks. Each rabbit was boosted twice at weeks 7 and 9 with the marginal veins of the ear. The Elsia experiments were performed at weeks 4,8, 9 and 10 to detect antibody titer and specificity, respectively.
Step 4, collecting rabbit anti-SPAG 8 polyclonal antibody serum
And 4.1, fixing four limbs of the rabbit in the supine position by using ropes, wherein the two upper limbs are crossed and arranged behind the head to be fixed, tying the upper jaw incisors of the rabbit by using a string and pulling the upper jaw incisors backwards, and fixing the rabbit head on the two upper limbs in a homeopathic manner.
And 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck by about 15cm from the suprasternal fossa to the lower jaw along the center of the neck, carefully separating subcutaneous tissues along the trachea after finding the trachea, and leading the far end of the trachea to the throat and the near end of the trachea to the sternocleidomastoid muscle.
And 4.3, showing a pulsating stiff artery below the trachea, carefully separating the carotid arteries on both sides, and fully dissociating.
And 4.4, sleeving two black silk threads into one side artery, separating the two black silk threads (one at the far end and the other at the near end), ligating the far end of the artery by using a silk thread, clamping the near end of the artery by using an artery clamp, shearing a small opening on the artery wall between the silk thread and the artery clamp by using an ophthalmic scissors, quickly inserting a prefabricated thin plastic hose, and quickly fixing the hose and the artery by using the near end silk thread to prevent the hose from dropping out and blood leakage.
And 4.5, slightly loosening the artery clamp, obliquely placing a 50ml centrifuge tube to receive arterial blood ejected from the blood-letting tube until no blood drips out, treating the carotid artery on the other side by the same method to increase the blood-letting amount, and pressing the heart when the blood-letting flow is slow to increase the blood-letting amount. 75ml of SPAG8 rabbit blood was obtained by this method.
Step 4.6 isolation and storage of Rabbit antiserum Rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first serum aspiration, the serum was centrifuged at 12000 rpm at 4 ℃ for 15min, and the serum aspirated for the second time. And finally obtaining about 50ml of SPAG8 antibody serum, subpackaging by using a 15ml centrifuge tube, respectively adding 0.01% NaN3 by volume, and storing in a refrigerator at the temperature of-20 ℃.
Step 5, separating and purifying the antibody by a saturated ammonium sulfate method
At 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 a saturated ammonium sulfate solution pH7.0 was added dropwise with gentle shaking.
At step 5.2, when the ammonium sulfate solution reached 50% saturation, the solution was left at 4 ℃ overnight.
And 5.3, placing the solution at 4 ℃, centrifuging for 20min at 10000 r/min, and removing supernatant to obtain globulin precipitate.
After dissolving the precipitate in 3ml of 0.01M pH7.4PBS at 4 ℃ in step 5.4, 1.5ml of a saturated ammonium sulfate solution was added dropwise (to bring the ammonium sulfate solution to 33% saturation), and the precipitate was allowed to settle for 30 min. This was repeated twice.
Centrifuging at 10000 rpm for 10min at 4 ℃ in the step 5.5, and removing the supernatant to obtain the antibody IgG precipitate.
At step 5.6, the supernatant was discarded, and 3ml of 0.01M pH7.4PBS was added to dissolve the antibody IgG precipitate, and the mixture was packed into a dialysis bag.
At step 5.7, the dialysis bag was dialyzed at 4 ℃ against 0.01M PBS solution (pH 7.4). During the period, the solution was changed several times until the external dialysate had no yellow change. 8) And (3) taking a sample in the dialysis bag, drying in vacuum, taking a small amount of the sample, diluting by proper times, and then determining the protein content.
Step 6, enzyme-linked immunosorbent assay for identifying the binding potency of the serum SPAG8 antibody and the natural human SPAG8 protein step 6.1, and preparation of human sperm membrane protein soluble antigen
After abstinence for 3-5 days in step 6.1.1, semen is collected by masturbation. After the sperms are 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 of a grade of more than 25 percent, or the a + b grade of more than 50 percent for later use; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form a two-layer Percoll density gradient separation column; 4ml of the ready-to-use semen were carefully added to the centrifuge tube, and a clear interface was visible. Centrifuging at 3000 rpm for 15min, carefully sucking sperm layer, adding into 2ml PBS solution, centrifuging at 4 deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4 deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Obtaining a clean sperm cell precipitate, fully mixing the precipitate with about 3-5ml PBS, and transferring the mixture into a 15ml sterile centrifuge tube to obtain a sperm cell suspension.
And 6.1.2, breaking the cell membrane of the sperm by using an ultrasonic cell disruptor, and fully exposing the antigen sites by using membrane protein. The method comprises the following steps: a15 ml centrifuge tube containing sperm cells (3-5 ml) was placed in crushed ice with the top cap open and the ultrasonic cell disruptor probe placed in solution with intensity adjusted to 70% for 6min (disruption for 5s, pause for 5s to prevent over-temperature antigen denaturation and foam generation).
And 6.1.3, centrifuging the solution (4 ℃, 12000 r/min), discarding the precipitate, taking the supernatant (sperm soluble membrane protein antigen), and storing at 4 ℃. Repeating the above steps, collecting the soluble antigens obtained in different times, and storing in a-80 deg.C refrigerator.
6.2, coating, namely, diluting the human sperm membrane protein antigen to 50ug/ml, dripping the diluted human sperm membrane protein antigen into a plurality of small holes of a 96-well plate, and keeping the diluted human sperm membrane protein antigen at 100 mu L/hole overnight at 4 ℃.
And 6.3, sealing: the soluble antigen in the wells was discarded, 150. mu.L/well of 5% skim milk powder (blocking solution) was added, and incubation was carried out at 37 ℃ for 40 min.
And 6.4, washing, namely adding 200ul of washing solution (PBST) per hole, washing for three times, and standing for 3min each time.
And 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of the serum antibody to be detected with different dilutions, and standing at 37 ℃ for 40 min.
And 6.6, washing, namely adding PBST into the small holes, keeping the small holes at 200 ul/hole, standing for 3min, discarding the washing liquid in the holes, repeating the steps for three times, and finally fully throwing the washing liquid in the holes.
Step 6.7, enzyme-labeled antibody (secondary antibody) is added, and HRP-labeled goat anti-rabbit secondary antibody is diluted at a ratio of 1:10000, added to 100. mu.L/well, and left at 37 ℃ for 40 min.
And 6.8, washing, namely washing three times by PBST for 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, adding 1M H2SO4, 50 μ L/well.
And 6.11, measuring by using an enzyme-linked immunosorbent assay (ELISA) instrument to determine the OD 490nm value. After ten times of immunization, the final 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 the highest; (FIG. 5).
7, positioning the SPAG8 protein distribution on the surface of the sperm by cell immunofluorescence staining
7.1, collecting human sperms and neutrophils: collecting semen of healthy people by masturbation, completely liquefying at 37 deg.C, centrifuging at 2000 rpm for 10min to collect spermatid, 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 0.01MPH7.4 PBS. Counting with a blood counting chamber, adjusting the concentration of sperm and neutrophil to 2 × 106Per ml;
step 7.2, sperm smear: uniformly dripping 0.5ml of PBS solution containing 5 x 106 sperms on a polylysine coated glass slide, and naturally drying at room temperature to form a sperm smear;
7.3, washing with 0.01MPH7.4PBS for 3 times, each time for 5 minutes;
7.4, covering the mixture with 4 percent paraformaldehyde for fixing for 15 minutes at room temperature, and keeping the mixture out of the sun;
7.5, washing with 0.01MPH7.4PB PBS buffer solution for 3 times, each time for 5 minutes;
step 7.6, 0.25% Triton X-100 (permeant) covering the cells for 5-7 minutes (note: this step can be omitted without adding permeant);
7.7, washing for three times with 0.01MPH7.4PBS for 5 minutes each time;
7.8, sealing the goat serum at room temperature for 40 minutes;
7.9, preparing a primary antibody, diluting the SPAG8 to 200ug/ml, and keeping the wet box at 4 ℃ away from light overnight;
7.10, washing with PBS for three times, 5 minutes each time;
7.11, preparing a fluorescence labeled secondary antibody (Ab150079) Goat anti-rabbitIgG (H & L), diluting the mixture with PBS according to a ratio of 1:100, and storing the mixture in a dark place; .
7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (keeping out of light);
7.13, washing three times with 4 ℃ PBS, each time for 15 minutes (protected from light);
step 7.14, staining the nuclei with DAPI, and completely covering the cells (keeping out of the sun);
at step 7.15, the staining result was observed by using a confocal microscope, and cells showing high-intensity red fluorescence (emission wavelength: 647nm) were observed as a visual field, and photographed. Immunofluorescent staining of human sperm and neutrophil smears showed that IgG antibodies were not stained in human sperm (fig. 6); the SPAG8 protein was mainly distributed in the middle of the neck and tail of sperm, and part of the protein was distributed in the tail of sperm, and the neutrophil was not stained (FIG. 7);
Step 8.1, collecting human normal mature sperms and neutrophils: collecting semen of healthy people by masturbation, completely liquefying at 37 deg.C, centrifuging at 2000 rpm for 10min to collect spermatid, 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 0.01MPH7.4 PBS. Counting with a blood counting chamber, adjusting the concentration of sperm and neutrophil to 2 × 106Per ml;
step 8.2, antibody and sperm co-incubation: respectively and uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of 2mg/ml SPAG8 antibody solution, incubating for 30 minutes at 37 ℃, washing and centrifuging for 3 times by using 0.01MPH7.4PBS, and fixing the volume to 10 ml; the subsequent operation steps are the same as the above steps 7.2 to 7.15; the SPAG8 antibody and sperm co-incubation experiment can observe the expression condition of the antibody combined with the SPAG8 protein of the motile sperm, the co-incubation experiment shows that the antibody generates fluorescence at the corresponding part of the sperm, and the result shows that the antigenic determinant of the SPAG8 protein is positioned at the outer side of the sperm cell membrane and can be directly combined with the antibody under the sperm living state (figure 8).
Claims (8)
1. The human sperm membrane protein SPAG8 specific polypeptide is characterized in that antigen binding sites are widely distributed on the outer membranes of the neck, the middle section and the tail of a human sperm, and the amino acid sequence of the polypeptide is as follows: GVSNIRTLDTPFRKNC are provided.
2. A method for preparing an antibody of anti-human sperm membrane protein SPAG8 is characterized by comprising the following steps:
1) and the analysis and design of the specific epitope of human sperm membrane protein SPAG 8;
2) human sperm membrane protein SPAG8 specific polypeptide antigen synthesis and carrier protein coupling purification;
3) preparing artificial immunogen immune animals;
4) collecting antibody serum;
5) separating and purifying the anti-human sperm membrane protein SPAG8IgG antibody by a saturated ammonium sulfate method;
6) enzyme linked immunosorbent assay is carried out to identify the binding titer of the serum antibody and the natural human SPAG8 protein;
7) and positioning the SPAG8 protein distribution on the surface of the sperm by using cell immunofluorescence staining.
3. The method for producing antibody according to claim 2, wherein the extracellular specific epitope sequence determined by the analysis and design of the specific epitope is a peptide fragment from the 422 th to the 437 th amino acids in the amino acid sequence of SPAG8, which is a human sperm membrane protein.
4. The method of claim 2, wherein the carrier protein is Keyhole Limpet Hemocyanin (KLH).
5. The method of claim 2, wherein the artificial immunogen is KLH-polypeptide mixed with complete or incomplete freund's adjuvant.
6. The method of claim 2, wherein the carrier protein is purified by coupling to synthesize the antigen polypeptide, and the amino acid sulfhydryl of the polypeptide is covalently linked with the amino acid sulfhydryl of the carrier protein by using a cross-linking agent, and the antibody is purified by using a chromatographic column.
7. The method of claim 2, wherein the immune animal is injected subcutaneously at multiple sites on the back of the experimental animal, and the serum titer of the immune animal is more than 1: 3200.
8. the method of claim 2, wherein the isolated and purified antibody is an IgG antibody having a high purity from the serum of the antibody by ammonium sulfate precipitation and protein affinity purification.
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|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114395028A (en) * | 2022-02-09 | 2022-04-26 | 天津市泌尿外科研究所 | Preparation method of human seminal membrane protein SPACA1 specific antigen coupling protein and antibody |
| CN114395028B (en) * | 2022-02-09 | 2024-01-30 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody |
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