CN114395028A - Preparation method of human seminal membrane protein SPACA1 specific antigen coupling protein and antibody - Google Patents
Preparation method of human seminal membrane protein SPACA1 specific antigen coupling protein and antibody Download PDFInfo
- Publication number
- CN114395028A CN114395028A CN202210120812.4A CN202210120812A CN114395028A CN 114395028 A CN114395028 A CN 114395028A CN 202210120812 A CN202210120812 A CN 202210120812A CN 114395028 A CN114395028 A CN 114395028A
- Authority
- CN
- China
- Prior art keywords
- protein
- spaca1
- antibody
- membrane protein
- human sperm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 101000637360 Homo sapiens Sperm acrosome membrane-associated protein 1 Proteins 0.000 title claims abstract description 88
- 102100032143 Sperm acrosome membrane-associated protein 1 Human genes 0.000 title claims abstract description 82
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 55
- 239000000427 antigen Substances 0.000 title claims abstract description 55
- 102000036639 antigens Human genes 0.000 title claims abstract description 55
- 108091007433 antigens Proteins 0.000 title claims abstract description 55
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 54
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 40
- 230000008878 coupling Effects 0.000 title claims abstract description 14
- 238000010168 coupling process Methods 0.000 title claims abstract description 14
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 37
- 229920001184 polypeptide Polymers 0.000 claims abstract description 30
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000002965 ELISA Methods 0.000 claims abstract description 7
- 230000000694 effects Effects 0.000 claims abstract description 7
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 6
- 235000018102 proteins Nutrition 0.000 claims description 38
- 210000002966 serum Anatomy 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 23
- 150000001413 amino acids Chemical class 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 17
- 235000001014 amino acid Nutrition 0.000 claims description 16
- 238000004458 analytical method Methods 0.000 claims description 15
- 241001465754 Metazoa Species 0.000 claims description 14
- 239000002671 adjuvant Substances 0.000 claims description 13
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 10
- 230000002163 immunogen Effects 0.000 claims description 8
- 238000009739 binding Methods 0.000 claims description 7
- 230000027455 binding Effects 0.000 claims description 6
- 102000056733 human SPACA1 Human genes 0.000 claims description 5
- 230000000890 antigenic effect Effects 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 4
- 238000003125 immunofluorescent labeling Methods 0.000 claims description 4
- 238000009826 distribution Methods 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 238000001261 affinity purification Methods 0.000 claims 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims 1
- 238000010171 animal model Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 23
- 230000006870 function Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 108010078791 Carrier Proteins Proteins 0.000 abstract description 2
- 102000014914 Carrier Proteins Human genes 0.000 abstract description 2
- 229960000074 biopharmaceutical Drugs 0.000 abstract description 2
- 238000004132 cross linking Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 230000036039 immunity Effects 0.000 abstract 1
- 238000001727 in vivo Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 47
- 238000005406 washing Methods 0.000 description 34
- 210000001367 artery Anatomy 0.000 description 18
- 239000002244 precipitate Substances 0.000 description 18
- 210000000440 neutrophil Anatomy 0.000 description 17
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 229940024606 amino acid Drugs 0.000 description 13
- 238000002156 mixing Methods 0.000 description 11
- 210000000170 cell membrane Anatomy 0.000 description 10
- 210000000582 semen Anatomy 0.000 description 10
- 210000003437 trachea Anatomy 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 230000003053 immunization Effects 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 238000007865 diluting Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000011534 incubation Methods 0.000 description 7
- 208000000509 infertility Diseases 0.000 description 7
- 230000036512 infertility Effects 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 241000283707 Capra Species 0.000 description 6
- 206010070834 Sensitisation Diseases 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000008313 sensitization Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 230000000469 anti-sperm effect Effects 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 210000001715 carotid artery Anatomy 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 235000013601 eggs Nutrition 0.000 description 4
- 238000004945 emulsification Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 210000004283 incisor Anatomy 0.000 description 4
- 208000021267 infertility disease Diseases 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 238000003825 pressing Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 210000001364 upper extremity Anatomy 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 231100000535 infertility Toxicity 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 210000004340 zona pellucida Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000030120 acrosome reaction Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 210000004013 groin Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000001632 homeopathic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 206010033675 panniculitis Diseases 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000009612 semen analysis Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 230000019100 sperm motility Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 210000004304 subcutaneous tissue Anatomy 0.000 description 2
- 235000011149 sulphuric acid Nutrition 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 208000007466 Male Infertility Diseases 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 230000021183 acrosome assembly Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 210000004517 glycocalyx Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 230000021595 spermatogenesis Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Pregnancy & Childbirth (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method for preparing a human sperm membrane protein SPACA1 specific antigen coupling protein and an antibody. The N-terminal specific polypeptide amino acid sequence is as follows: NYAPPETEDVSNRNC are provided. The preparation of the anti-human sperm membrane protein SPACA1 antibody comprises the following steps: (1) analyzing and designing 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 SPACA1 antibody prepared by the invention has high titer, strong affinity and good specificity, can be specifically combined with the epitope of human sperm membrane protein SPACA1 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 SPACA1, and has good application prospects in the fields of targeted tracing, biological pharmacy, precise treatment and the like.
Background
Capacitation of sperm is an important step in the process of amphoterism, where the sperm cell membrane is transformed from a non-fertile state to a fertile state. After the sperm is capacitated, the sperm membrane protein, glycoprotein, lipid component and the sperm plasma membrane channel are changed in various molecular structures, so that on one hand, the loss of the sperm head plasma membrane component and the change of the sperm tail cell membrane occur, and the sperm super-activation occurs; sperm on the other hand feed back and generate Acrosome Reactions (AR) on signals provided by the egg cells and their associated structures (granulocytes, zona pellucida). The processes of activating acrosome receptor, fusing acrosome membrane with sperm cell plasma membrane, releasing hydrolase in acrosome, hydrolyzing egg cell coat (zona pellucida) and the like occur, and finally the sperm-oocyte fusion is promoted. These receptor, carbohydrate or protein molecules on the sperm acrosome region can act as antigens to directly generate antisperm antibodies (AsAb), thereby interfering with sperm-egg fusion and causing male infertility. Because of lack of research on specific antigen sites and antibodies of sperm acrosome membrane proteins, the action mechanism of male immune infertility caused by the AsAb is not clear, so by means of interdisciplinary application of technologies such as molecular biology, bioinformatics, high-throughput sequencing and the like, the action mechanism of the specific antigen sites of sperm acrosome zone, neck and tail sperm surface membrane proteins in the process of amphoterism is further disclosed, a series of scientific problems such as construction of a specific immune infertility targeted treatment method and the like are established, and the method is a great demand for promoting healthy fertility and building population safety.
(1) Function of SPACA 1: the human sperm acrosome membrane-associated protein 1 (SPACA 1) gene is located in the sense chain of 6q15DNA, the length is 19,798bp, 8 exons are provided, and a conserved structural domain is provided, and the mRNA of the gene has two subtypes: XM _011536160.2 and XM _ 017011335.1. The SPACA1 protein product has 294 amino acids and a molecular weight of 32,143Da, is a single-transmembrane type I membrane protein and is mainly located in a cell membrane. SPACA1 was detected in the acrosomes at all stages of sperm cell development during spermatogenesis, but not before acrosome formation. The protein localizes to the inner and outer membranes of the entire acrosome of sperm cells and mature sperm. There are studies showing that SPACA1 plays a major role in acrosome morphogenesis and sperm-egg binding and fusion.
(2) Information on SPACA1 antibody product: through search, the SPACA1 protein polyclonal antibody is on the market, but the epitope of the antibody is different and can only be detected in vitro.
(3) Function of SPACA1 antibody: the SPACA1 antibody has relevance to idiopathic infertility cases, and the protein expressed by the gene is firstly identified by antisperm antibody of sterile male. In vitro experiments show that SPACA1 plays a physiological role in the combination and fusion of sperms and egg membranes, and antibodies generated by SPACA1 recombinant protein can inhibit the combination of human sperms and eggs of hamster with zona pellucida, thereby blocking in vitro fertilization. The recombinant SPACA1 antigen has strong immunological binding reaction with the serum of sterile male with positive antisperm antibody, and it is suggested that SPACA1 may be the antigen causing immunological sterility. Animal experiments show that the SPACA1 protein plays an important role in the formation of sperm head, and mature sperm of a SPACA1 knockout mouse is round-head sperm lacking acrosome and can cause sterility.
Disclosure of Invention
The invention aims at the defects of an AsAb preparation method and a research tool for immune sterility mechanism research, and provides a preparation method of a human spermatic membrane protein SPACA1 specific polypeptide, an antigen coupling protein and an antibody, which is different from an in vitro experiment detection antibody, and the anti-SPACA 1 antibody prepared by the polypeptide can specifically identify natural human SPACA1 protein in tissues or cells through immunofluorescence and enzyme-linked immunosorbent assay on one hand, and can perform anti-sperm antibody mediated immune sterility mechanism research on the other hand by means of specific surface antigenic sites of SPACA1, and can also perform targeted tracing, biopharmaceutical and precise treatment on the other hand.
Technical scheme of the invention
An N-terminal human sperm membrane protein SPACA1 specific polypeptide, the antigen binding site of which is positioned on the outer membrane of a human sperm acrosome region, the polypeptide sequence is a peptide segment from 61 st to 74 th amino acids in the amino acid sequence of human sperm membrane protein SPACA1, and cysteine (C) is added at the C terminal, the amino acid sequence of the polypeptide is: NYAPPETEDVSNRNC are provided.
A human sperm membrane protein SPACA1 specific antigen coupling protein is a mixture of synthetic antigen polypeptide NYAPPETEDVSNRNC and Keyhole Limpet Hemocyanin (KLH) amino covalently cross-linked polypeptide amino acid sulfhydryl and Keyhole Limpet Hemocyanin (KLH) amino by a cross-linking agent.
The invention also provides a preparation method of the antibody of the anti-human sperm membrane protein SPACA1, 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, predicting a transmembrane region of a membrane protein on line by using TMHMM;
logging in a TMHMM main page, submitting a FASTA format SPACA1 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, using SignalP 4.1 Server to carry out predictive analysis of signal peptide on membrane protein;
logging in a SignalP 4.1 Server 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, performing prediction analysis on hydrophobicity of the membrane protein 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'; 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 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%.
2.2, synthesizing specific antigen coupling protein by EDC coupling;
preparing keyhole limpet hemocyanin KLH, synthetic polypeptide and EDC into solutions respectively, and mixing the polypeptide solutions according to the weight ratio of 1: 1-2, and immediately adding the solution into a keyhole limpet hemocyanin KLH solution according to the mass ratio of 1: EDC solution was added to the mcKLH peptide solution at a ratio of 4-5, and the residue of EDC was removed after 2 hours of reaction at room temperature.
Step 2.3, purifying the specific antigen-conjugated protein 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 3.1, purchasing SPF-grade New Zealand white rabbits with 2.0KG weight is divided into experimental group and control group, and normal serum is prepared from animal marginal vein blood of 0.5ml before immune experiment.
3.2, wrapping the antigen by using an adjuvant: before each immunization, 600ug of the specific antigen-coupled protein complex 400-400 was inhaled into a 2.5ml sterile syringe, the other 2.5ml syringe was inhaled into the same amount of complete Freund's adjuvant or Freund's incomplete adjuvant, the two syringes were connected by a sterile plastic hose and repeatedly pumped until complete emulsification (i.e., no diffusion of one drop into cold water, i.e., complete emulsification of water-in-oil) was achieved, and then the mixture was used for immunization.
3.3, animal sensitization: 400mg of specific antigen-conjugated protein complex and complete Freund's adjuvant in a volume ratio of 1:1, mixing, performing the 2 nd step, fully mixing, performing animal sensitization, and injecting into the animal body, neck and back by multipoint method (each point is about 200ul) for increasing sensitization effect.
Step 3.4 antigen was boosted weekly with the same antigen dose and cross-emulsified with Freund's incomplete adjuvant and Freund's complete adjuvant, 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.
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. Finally, about 50ml of SPACA1 antibody serum is obtained, and is subpackaged by a 15ml centrifuge tube, 0.01 percent NaN3 by volume is respectively added, and the mixture is stored in a refrigerator at the temperature of 20 ℃ below zero.
5, separating and purifying the anti-human sperm membrane protein SPACA1IgG 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.
and 6.1, preparing the 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 SPACA1 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 SPACA1 to 150ug/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.
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 by using a blood counting chamber, and adjusting the concentration of both sperms and neutrophils to be 2 multiplied by 106/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 SPACA1 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-SPACA 1 antibody prepared by the invention not only can have stable specific binding capacity with the fixed denatured SPACA1 protein antigen site, but also can be specifically bound with the sperm acrosome membrane SPACA1 protein antigen site which keeps the cell integrity in a physiological state, thereby obtaining the specific 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. As the SPACA1 protein is widely distributed on the outer membrane of the sperm acrosome region, the antibody prepared by the invention has good application prospect in the fields of targeted tracing of sperm, biological pharmacy, precise treatment and the like.
Drawings
Figure 1 amino acid sequence of human SPACA1 and amino acid sequence of specific SPACA1 epitope.
FIG. 2TMHMM2.0 predicts that the transmembrane domain of the SPACA1 protein is at position 217 and 239, and the amino acids at positions 1-216 are outside the cell membrane.
Figure 3SignalP 4.1 predicts SPACA1 to be a secreted protein with sequence cleavage site at position 30, Smax ═ 0.967, Ymax ═ 0.840.
FIG. 4ExPASY-ProtScale predicts 6 forward peaks in the sequence of SPACA1 protein and the highest score for amino acid 238 (ILE, isoleucine) indicating the most hydrophobic amino acid at this site; the 5 distinct negative peaks, the amino acid at position 53 (Glu ) had the lowest score, indicating the most hydrophilic amino acid at this position.
FIG. 5ELSIA determination of the potency of the SPACA1 antibody, (A) the fourth week antibody potency was 1: 1600; (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. 7SPACA1 protein was mainly concentrated in the acrosome region of sperm head and no staining was seen in neutrophils.
FIG. 8 shows that the SPACA1 protein antibody against human sperm membrane can not directly bind with the antibody outside the sperm membrane, and the immunofluorescence results of the co-incubation of the SPACA1 protein and the sperm show that the antigenic determinant is positioned in the sperm acrosome cavity under the living state of the sperm.
Detailed Description
Example 1:
a preparation method of a human sperm membrane protein SPACA1 specific antigen coupling protein and an antibody specifically comprises the following steps:
the amino acid sequence of the SPACA1 protein is subjected to epitope analysis by using biological information software, transmembrane fragment, polypeptide activity, hydrophilicity, antigenicity and other indexes are mainly evaluated, and by combining actual experience of preparing antibodies in the past, 15 amino acids at positions 61-74 of the SPACA1 protein are finally determined, a cysteine is modified at the C end of the polypeptide, and the synthetic polypeptide amino acid sequence is NYAPPETEDVSNRNC (figure 1).
Step 1.1, using TMHMM to predict the transmembrane region of human sperm membrane protein SPACA1 on line;
logging in a TMHMM main page, submitting a FASTA format SPACA1 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 the transmembrane region of the SPACA1 protein sequence is positioned at 217-239 bit, and 1-216 bits are positioned in the inner membrane of the acrosome region; (FIG. 2).
Step 1.2, using SignalP 4.1 Server to carry out prediction analysis of signal peptide on human sperm membrane protein SPACA 1;
logging in a SignalP 4.1 Server 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 according to a submit key and checking a prediction analysis result, wherein the cleavage site of the SPACA1 protein sequence is 30 bits, Smax is 0.967, and Ymax is 0.840; (FIG. 3).
Step 1.3, performing hydrophobicity prediction analysis on human sperm membrane protein SPACA1 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 predictive analysis is submitted according to 'Submit', the SPACA1 protein sequence has 6 forward peaks, and the amino acid (ILE, isoleucine) at the 238 th position has the highest score, which indicates that the amino acid at the position has the strongest hydrophobicity. The 5 distinct negative peaks, with the lowest score for amino acid at position 53 (Glu, glutamate), indicate the most hydrophilic amino acid at this position (figure 4).
step 2.1, synthesis of SPACA1 artificial polypeptide;
shanghai Huada Gene company adopts an automatic polypeptide synthesizer to synthesize and purify 10mg of SPACA1(16 peptide), and adopts mass spectrometry and high performance liquid chromatography to identify the purity, wherein the purity is more than 95%.
2.2, combining the human sperm membrane protein SPACA1 specific antigen coupling protein by EDC coupling;
KLH was prepared as a 10mg/ml solution 1.25ml, SPACA1(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, 250. mu.l of this solution was immediately added to the mcKLH peptide solution, and the EDC residue was removed after 2 hours reaction at room temperature.
2.3, purifying a human sperm membrane protein SPACA1 specific antigen-coupled protein 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 human sperm membrane protein SPACA1 specific antigen-coupled protein complex into a 25ml Purification Buffer Salts elution column, standing for 2 minutes, eluting the column by using 5ml of Purification Buffer solution, collecting the eluate, measuring the absorbance at 280nm by using an ultraviolet visible absorption spectrum, and calculating the content of the polypeptide-KLH complex according to the 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 ℃.
at step 3.1, 4 new Zealand white rabbits of SPF grade with a weight of 2.0KG were purchased. The normal sera were prepared from 0.5ml of blood drawn from the ear margin of animals before the immunization experiment, and divided into SPACA1 group and control group.
3.2, wrapping the antigen by using an adjuvant: before each immunization, 600ug of the human sperm membrane protein SPACA1 specific antigen-coupled protein complex is inhaled into a 2.5ml sterile syringe, the other 2.5ml syringe is used for inhaling 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 longer diffusing, namely reaching the complete emulsification degree of water-in-oil), and the immune system can be used for immunization.
3.3, animal sensitization: 400mg of human sperm membrane protein SPACA1 specific antigen-coupled protein complex and complete Freund's adjuvant in a volume ratio of 1:1, mixing, performing the 2 nd step, fully mixing, performing animal sensitization, and injecting into the animal body, neck and back by multipoint method (each point is about 200ul) for increasing sensitization effect.
Step 3.4 antigen was boosted weekly with the same antigen dose and cross-emulsified with Freund's incomplete adjuvant and Freund's complete adjuvant, 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. 5-10ml of blood was collected from the 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.
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. This procedure gave 70ml of SPACA1 rabbit blood.
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. Finally, about 50ml of SPACA1 antibody serum is obtained, and is subpackaged by a 15ml centrifuge tube, 0.01 percent NaN3 by volume is respectively added, and the mixture is stored in a refrigerator at the temperature of 20 ℃ below zero.
5, 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.
6.1, preparing a 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.
Step 6.11, measurement: OD 490nm was measured by a microplate reader. After ten immunizations, the final titers of the SPACA1 antibody at the fourth, eighth, ninth and tenth weeks were 1:1600, 1:3200 and 1:3200, respectively, and the antibody OD value was highest at ten weeks (FIG. 5).
7, positioning the SPACA1 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 SPACA1 to 150ug/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); SPACA1 protein was mainly concentrated in the acrosome region of sperm head, and no staining was seen in neutrophils (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 SPACA1 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 SPACA1 antibody and sperm co-incubation experiment can observe the expression condition of antibody-bound motile sperm SPACA1 protein, and the co-incubation experiment shows that the SPACA1 protein has an antigenic determinant positioned in the apical body cavity of the sperm and can not be directly bound with the antibody outside the sperm cell membrane in the sperm living state. (FIG. 8).
Claims (8)
1. An N-terminal human sperm membrane protein SPACA1 specific polypeptide is characterized in that an antigen binding site is positioned on the outer membrane of a human sperm acrosome region, the polypeptide sequence is a peptide segment from 61 st to 74 th amino acids in the amino acid sequence of human sperm membrane protein SPACA1, cysteine (C) is added at the C terminal, and the amino acid sequence of the polypeptide is as follows: NYAPPETEDVSNRNC are provided.
2. A human sperm membrane protein SPACA1 specific antigen coupling protein is characterized in that a mixture of polypeptide amino acid sulfhydryl and Keyhole Limpet Hemocyanin (KLH) amino is covalently crosslinked by a crosslinking agent for synthesizing an antigen polypeptide NYAPPETEDVSNRNC.
3. A preparation method of an antibody of anti-human sperm membrane protein SPACA1 is characterized by comprising the following steps:
1) and the analysis and design of the specific epitope of human sperm membrane protein SPACA 1;
2) preparing and purifying a human sperm membrane protein SPACA1 specific antigen coupling protein;
3) preparing artificial immunogen immune animals;
4) collecting antibody serum;
5) separating and purifying the anti-human sperm membrane protein SPACA1IgG 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 SPACA1 protein;
7) and positioning the SPACA1 protein distribution on the surface of the sperm by using cell immunofluorescence staining.
4. The method for producing an antibody according to claim 3, wherein the analysis and design of the specific epitope is epitope analysis of an amino acid sequence of a protein using bioinformatics software, mainly evaluating transmembrane segment, polypeptide activity, hydrophilicity and antigenic index; the determined extracellular specific antigen epitope sequence is a peptide segment from 61 st to 74 th amino acids in the amino acid sequence of human sperm membrane protein SPACA1, and cysteine (C) is added at the C end, and the amino acid sequence of the polypeptide is as follows: NYAPPETEDVSNRNC are provided.
5. The method of claim 3, wherein the conjugated protein is Keyhole Limpet Hemocyanin (KLH).
6. The method for preparing antibody according to claim 3, wherein the artificial immunogen is the human sperm membrane protein SPACA1 specific antigen-coupled protein of claim 2 mixed with Freund's complete or incomplete adjuvant to immunize animals.
7. The method of claim 3, wherein the immunized animal is injected subcutaneously at multiple sites on the back of the experimental animal, and the serum titer of the antibody is more than 1: 3200.
8. the method of claim 3, wherein the isolated and purified anti-human sperm membrane protein SPACA1IgG antibody is an IgG antibody that can be obtained from antibody serum at high purity by ammonium sulfate precipitation and protein affinity purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210120812.4A CN114395028B (en) | 2022-02-09 | 2022-02-09 | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210120812.4A CN114395028B (en) | 2022-02-09 | 2022-02-09 | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114395028A true CN114395028A (en) | 2022-04-26 |
| CN114395028B CN114395028B (en) | 2024-01-30 |
Family
ID=81232411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210120812.4A Active CN114395028B (en) | 2022-02-09 | 2022-02-09 | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114395028B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030087375A1 (en) * | 2000-01-19 | 2003-05-08 | Zhonglin Hao | Sperm specific proteins |
| WO2010129310A1 (en) * | 2009-04-27 | 2010-11-11 | Roswell Park Cancer Institute | Reagents and methods for producing bioactive secreted peptides |
| US20120122244A1 (en) * | 2009-06-25 | 2012-05-17 | National University Corporation Chiba University | Method Of Identifying Compounds That Inhibit Fertilization |
| CN106932567A (en) * | 2017-02-27 | 2017-07-07 | 天津市泌尿外科研究所 | A kind of preparation method of the immune targeted nano gold of recombinant protein A mediation |
| CN113896769A (en) * | 2021-11-16 | 2022-01-07 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody |
-
2022
- 2022-02-09 CN CN202210120812.4A patent/CN114395028B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030087375A1 (en) * | 2000-01-19 | 2003-05-08 | Zhonglin Hao | Sperm specific proteins |
| WO2010129310A1 (en) * | 2009-04-27 | 2010-11-11 | Roswell Park Cancer Institute | Reagents and methods for producing bioactive secreted peptides |
| US20120122244A1 (en) * | 2009-06-25 | 2012-05-17 | National University Corporation Chiba University | Method Of Identifying Compounds That Inhibit Fertilization |
| CN106932567A (en) * | 2017-02-27 | 2017-07-07 | 天津市泌尿外科研究所 | A kind of preparation method of the immune targeted nano gold of recombinant protein A mediation |
| CN113896769A (en) * | 2021-11-16 | 2022-01-07 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG T等: "sperm acrosome membrane-associated protein 1 precursor [Homo sapiens]", GENBANK, pages 3 - 4 * |
| 李秋华;周泽荣;祝晓丽;谭颖;钟小英;姜荣华;宋革;: "精子表面透明带蛋白受体的分析与功能研究", 实用医院临床杂志, no. 01 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114395028B (en) | 2024-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2658491C2 (en) | HUMAN ANTIBODIES TO Fel d1 AND METHODS OF USE THEREOF | |
| CN110272490B (en) | Targeted CTLA-4 antibody, preparation method and application thereof | |
| CN109438576A (en) | A kind of preparation and its application of anti-human CD47 monoclonal antibody | |
| WO2022166806A1 (en) | Novel cd271-based antigenic epitope and application thereof | |
| CN113896769B (en) | Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody | |
| EP0142345A2 (en) | Anti-idiotypic vaccine employing anti-idiotypic monoclonal antibodies | |
| CN117074674B (en) | Colloidal gold test paper for detecting N-acetyl-beta-D glucosaminidase and preparation method thereof | |
| JPH01501793A (en) | Method for contraception by immunization against the zona pellucida | |
| CN114395028B (en) | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody | |
| WO2023155896A1 (en) | Use of methylation modification of lysine 162 in human pd-l1 protein in predicting sensitivity of malignant tumor immunotherapy | |
| CN114133442B (en) | Preparation method of human sperm membrane protein SPACA3 specific polypeptide and antibody | |
| CN107298697A (en) | Human PD-L1 protein Y123Site phosphorylation antibody and preparation method and application thereof | |
| CN114349846B (en) | Preparation method of human sperm membrane protein OR1D2 specific polypeptide and antibody | |
| CN109535255A (en) | A kind of anti-human CD26 antibody and its application in detection kit | |
| AU618024B2 (en) | Antibodies to angiogenin: immunotherapeutic agents | |
| EP0582450A2 (en) | Anti-oxytocin receptor antibodies and methods for their production | |
| Hirano et al. | Presence of immunoglobulin binding factor on human sperm surface as sperm coating antigen | |
| Campbell | Production and purification of antibodies | |
| CN113512106B (en) | Synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof | |
| JP4599527B2 (en) | Method for producing a hybridoma producing an antibody that recognizes the three-dimensional structure of a cell membrane protein | |
| CN114316053B (en) | VCE monoclonal antibody and preparation method thereof | |
| EP0653440B1 (en) | Monoclonal antibodies against hair follicle, hybridomas producing them | |
| CN116284370B (en) | Multimeric glycosylated hemoglobin monoclonal antibody and preparation method thereof | |
| CN117069853B (en) | Antibody targeting trastuzumab and application thereof | |
| Hayat | Antigens and antibodies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |