CN114395028B - Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody - Google Patents
Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody Download PDFInfo
- Publication number
- CN114395028B CN114395028B CN202210120812.4A CN202210120812A CN114395028B CN 114395028 B CN114395028 B CN 114395028B CN 202210120812 A CN202210120812 A CN 202210120812A CN 114395028 B CN114395028 B CN 114395028B
- Authority
- CN
- China
- Prior art keywords
- spaca1
- protein
- 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.)
- Active
Links
- 101000637360 Homo sapiens Sperm acrosome membrane-associated protein 1 Proteins 0.000 title claims abstract description 86
- 102100032143 Sperm acrosome membrane-associated protein 1 Human genes 0.000 title claims abstract description 81
- 239000000427 antigen Substances 0.000 title claims abstract description 59
- 102000036639 antigens Human genes 0.000 title claims abstract description 59
- 108091007433 antigens Proteins 0.000 title claims abstract description 59
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 54
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 48
- 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 16
- 238000010168 coupling process Methods 0.000 title claims abstract description 16
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 claims abstract description 32
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 32
- 238000004458 analytical method Methods 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 7
- 238000002965 ELISA Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 6
- 235000018102 proteins Nutrition 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 25
- 210000002966 serum Anatomy 0.000 claims description 24
- 150000001413 amino acids Chemical class 0.000 claims description 21
- 235000001014 amino acid Nutrition 0.000 claims description 16
- 239000002671 adjuvant Substances 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 238000002649 immunization Methods 0.000 claims description 11
- 230000003053 immunization Effects 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000002474 experimental method Methods 0.000 claims description 9
- 230000002163 immunogen Effects 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 238000009739 binding Methods 0.000 claims description 7
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 7
- 230000027455 binding Effects 0.000 claims description 6
- 102000056733 human SPACA1 Human genes 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 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
- 125000003277 amino group Chemical group 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims 2
- 238000001261 affinity purification Methods 0.000 claims 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 239000007929 subcutaneous injection Substances 0.000 claims 1
- 238000010254 subcutaneous injection Methods 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 24
- 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
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000004132 cross linking Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 101000618112 Homo sapiens Sperm-associated antigen 8 Proteins 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
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 37
- 238000005406 washing Methods 0.000 description 25
- 210000000440 neutrophil Anatomy 0.000 description 17
- 239000007788 liquid Substances 0.000 description 14
- 229940024606 amino acid Drugs 0.000 description 13
- 210000004369 blood Anatomy 0.000 description 13
- 239000008280 blood Substances 0.000 description 13
- 239000002244 precipitate Substances 0.000 description 12
- 210000000170 cell membrane Anatomy 0.000 description 11
- 210000001367 artery Anatomy 0.000 description 10
- 238000007865 diluting Methods 0.000 description 10
- 210000000582 semen Anatomy 0.000 description 10
- 238000000502 dialysis Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 208000000509 infertility Diseases 0.000 description 7
- 230000036512 infertility Effects 0.000 description 7
- 210000001715 carotid artery Anatomy 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 238000005520 cutting process Methods 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
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 210000003437 trachea Anatomy 0.000 description 6
- 238000004945 emulsification Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- 206010070834 Sensitisation Diseases 0.000 description 4
- 230000000469 anti-sperm effect Effects 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 238000004820 blood count Methods 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 235000013601 eggs Nutrition 0.000 description 4
- 238000011534 incubation Methods 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
- 229920003023 plastic Polymers 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 230000008313 sensitization Effects 0.000 description 4
- 230000019100 sperm motility Effects 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 210000001364 upper extremity Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000035558 fertility Effects 0.000 description 3
- 231100000535 infertility Toxicity 0.000 description 3
- 238000003825 pressing Methods 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
- 241000206602 Eukaryota Species 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
- 101000686985 Mouse mammary tumor virus (strain C3H) Protein PR73 Proteins 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
- 238000004847 absorption spectroscopy Methods 0.000 description 2
- 230000030120 acrosome reaction Effects 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 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
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000001632 homeopathic effect Effects 0.000 description 2
- 210000004283 incisor Anatomy 0.000 description 2
- 210000000494 inguinal canal Anatomy 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
- 238000004949 mass spectrometry Methods 0.000 description 2
- 210000001595 mastoid Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000011587 new zealand white rabbit Methods 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
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 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
- 230000008010 sperm capacitation Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 210000001562 sternum Anatomy 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
- 231100000617 superantigen Toxicity 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000008685 targeting 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
- 206010021929 Infertility male Diseases 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
- 241000699670 Mus sp. Species 0.000 description 1
- 240000007711 Peperomia pellucida Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 101150065144 SPACA1 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 101710119048 Sperm acrosome membrane-associated protein 1 Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000746 body region Anatomy 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
- 238000005516 engineering process Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 210000004517 glycocalyx Anatomy 0.000 description 1
- 210000002503 granulosa cell Anatomy 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000001939 inductive effect Effects 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
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000021595 spermatogenesis Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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 preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody. The N-terminal specific polypeptide has the amino acid sequence: NYAPPETEDVSNRNC. The preparation of the anti-human sperm membrane protein SPACA1 antibody comprises the following steps: (1) analysis and design of specific extracellular antigen epitope; (2) specific artificial polypeptide synthesis; (3) cross-linking the synthetic polypeptide with a carrier protein; (4) preparing a rabbit polyclonal antibody; and (5) collecting, separating and purifying to obtain the antibody. The specific anti-human sperm membrane protein SPACA1 antibody prepared by the invention has high titer, strong affinity and good specificity, can be specifically combined with the human sperm membrane protein SPACA1 epitope with natural activity in vivo and in vitro, and can be used for fluorescent immunity and enzyme-linked immunosorbent assay. The antibody not only provides an important tool for researching the characteristics, functions, expression profiles and related diseases of the human sperm membrane protein SPAG8, but also has application prospects in the fields of targeted tracking, bio-pharmacy, accurate treatment and the like.
Description
Technical Field
The invention relates to a polypeptide and a preparation method of an antibody thereof, wherein the antibody is an important tool for basic research of human sperm membrane protein SPACA1 protein, and has good application prospect in the fields of targeted tracking, bio-pharmacy, accurate treatment and the like.
Background
Sperm capacitation is an important step in the process of ampholytic reproduction, in which the sperm cell membrane is transformed from a non-fertility state to a fertility state. After sperm is capacitation, sperm membrane proteins, glycoprotein, lipid components and sperm plasma membrane channels change in various molecular structures, on one hand, loss of sperm head plasma membrane components occurs, tail cell membranes change, and sperm super-activation occurs; sperm on the other hand feed back signals provided by egg cells and their associated structures (granulosa cells, zona pellucida) and undergo the Acrosome Reaction (AR). Activation of the acrosome receptor, fusion of the acrosome membrane with the sperm cytoplasmic membrane, release of hydrolase in the acrosome, hydrolysis of the egg cell coat (zona pellucida), and the like occur, and sperm-oocyte fusion is finally promoted. These receptor, sugar or protein molecules on the acrosome region of sperm can act as antigens to directly produce anti-sperm antibodies (asabs), thereby preventing sperm-egg fusion and triggering male infertility. Because of the lack of research on specific antigenic sites and antibodies of the acrosome membrane proteins, the action mechanism of the AsAb for inducing male immune infertility is not clear, and therefore, by means of interdisciplinary application of technologies such as molecular biology, bioinformatics, high-throughput sequencing and the like, the action mechanism of the specific antigenic sites of the surface membrane proteins of the acrosome region, neck and tail sperm in the process of ampholytic reproduction is further revealed, and a series of scientific problems such as construction of specific immune infertility targeted therapy methods and the like are important requirements for promoting healthy fertility and constructing population safety.
(1) Function of SPACA 1: the human sperm acrosome membrane associated protein 1 (sperm acrosome membrane-associated protein 1, SPACA 1) gene is located on the 6q15DNA sense strand, is 19,798bp in length, has 8 exons, has a conserved domain, and 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, and is a single transmembrane type I membrane protein, and is mainly located in cell membranes. SPACA1 was detected in the acrosomes at all stages of sperm cell development during spermatogenesis, but was not found until acrosomes were formed. The protein localizes to the inner and outer membranes of the whole acrosomes of sperm cells and mature sperm. There are studies showing that SPACA1 plays a major role in acrosome morphology and sperm-egg binding and fusion.
(2) Information on SPACA1 antibody product: polyclonal antibodies to the SPACA1 protein are available on the market, but these antibodies differ in their epitopes and can only be detected in vitro.
(3) Function of SPACA1 antibody: the SPACA1 antibody has correlation with idiopathic infertility cases, and the protein expressed by the gene is recognized by an anti-sperm antibody of an infertility male at the earliest. In vitro experiments show that SPACA1 plays a physiological role in the combination and fusion of sperms and egg membranes, and antibodies generated by the SPACA1 recombinant protein can inhibit the combination of human sperms and hamster eggs with zona pellucida so as to block in vitro fertilization. The recombinant SPACA1 antigen has strong immune binding reaction with sterile male serum positive with anti-sperm antibodies, which suggests that SPACA1 may be an antigen causing immune sterility. Animal experiments show that SPACA1 protein plays an important role in the formation of sperm heads, and mature sperm of SPACA1 gene knockout mice are round head sperm lacking acrosome and can cause sterility.
Disclosure of Invention
The invention aims at overcoming the defects of an AsAb preparation method and a research tool which can be used for researching an immune sterility mechanism, and provides a preparation method of human sperm membrane protein SPACA1 specific polypeptide, antigen coupling protein and antibody, which is different from an in-vitro experiment detection antibody, and an 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 the one hand, and can research an immune sterility action mechanism mediated by an anti-sperm antibody by means of the specific surface antigen locus of SPACA1 on the other hand, and can also develop researches in the aspects of targeted tracing, bio-pharmacy, accurate treatment and the like.
The technical proposal of the invention
An N-terminal human sperm membrane protein SPACA1 specific polypeptide, wherein an antigen binding site is positioned on an outer membrane of a human sperm acrosome region, a polypeptide sequence is a peptide segment from 61 st to 74 th amino acids in an amino acid sequence of the human sperm membrane protein SPACA1, cysteine (C) is added at a C terminal, and the amino acid sequence of the polypeptide is as follows: NYAPPETEDVSNRNC.
A human sperm membrane protein SPACA1 specific antigen coupling protein is a mixture of synthesized antigen polypeptide NYAPPETEDVSNRNC by covalent crosslinking of polypeptide amino acid hydrophobic groups and Keyhole Limpet Hemocyanin (KLH) amino groups through a crosslinking agent.
The invention also provides a preparation method of the antibody for resisting the human sperm membrane protein SPACA1, which comprises the following steps:
step 1, analyzing and designing specific antigen epitope of human sperm membrane protein SPACA 1;
the amino acid sequence of the protein is subjected to antigen epitope analysis by utilizing biological information software, and indexes such as transmembrane fragments, polypeptide activity, hydrophilicity, antigenicity and the like are mainly evaluated.
Step 1.1, online predicting a transmembrane region of a membrane protein using TMHMM;
logging in a TMHMM main page, submitting a FASTA format SPACA1 protein sequence in a popup box of a 'select file' or pasting the sequence in a text box under 'OR by pasting sequence(s) in FASTA format'; out formats have three options, respectively, graphical output (existence); text output (no graphics); displaying proteins line by line (One line protein), and displaying a default option graphically; after the adjustment is finished, a submit key is pressed to submit and check the prediction analysis result.
Step 1.2, performing predictive analysis of signal peptides on membrane proteins by using SignalP 4.1 Server;
logging in a SignalP 4.1 Server main page, submitting a FASTA format protein sequence in a pop-up box of a 'selected file' or pasting the sequence in a text box below; parameter setting organosm group: selecting Eukaryotes; d-cutoff values: selecting Default (optimized for correlation); graphics output: selecting PNG (inline); output format: a Standard; method selection Input sequences may include TM regions. And after the parameter setting is finished, pressing a submit key to submit and check a prediction analysis result.
Step 1.3, predictive analysis of membrane protein hydrophobicity using ExpASY-ProtScale;
logging in an ExPASY-ProtScale main interface, and inputting a protein sequence ID number in a text box of 'Enter a UniProtKB/Swiss-Prot or UniProtKB/TrEMBL accession number (AC)'; or the FASTA format protein sequence is stuck to a text box of Or you can paste your own sequence in the box below; and selecting default setting, submitting and checking a prediction analysis result according to the 'subset' after the setting is finished.
Step 2, preparing and purifying the human sperm membrane protein SPACA1 specific antigen coupling protein;
step 2.1, synthesizing artificial polypeptide;
and synthesizing and purifying by using an automatic polypeptide synthesizer, and identifying the purity by using mass spectrometry and high performance liquid chromatography, wherein the purity is more than 95%.
Step 2.2, 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 a ratio of 1:1-2 into a carrier keyhole limpet hemocyanin KLH solution, and immediately mixing the polypeptide with the synthesized polypeptide according to the mass ratio of 1:4-5 to mcKLH peptide solution, and removing the residue of EDC after 2 hours of reaction at room temperature.
Step 2.3, purifying the specific antigen-coupled protein complex;
60ml of ultra-pure water degassed by ultrasonic vibration is added into a Purification Buffer Salts bottle to dissolve the content, a desalting column is used for filtering every 0.5ml of sample, 0.5ml of synthesized polypeptide-KLH complex is gently dripped into 25ml of Purification Buffer Salts eluting column, standing is carried out for 2 minutes, 5ml of purification buffer is taken for eluting the column, the absorbance of the eluent is detected at 280nm by ultraviolet-visible absorption spectrometry, and the content of the polypeptide-KLH complex is calculated according to the absorption peak. If the immunogen needs to be stored for a longer period of time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing an artificial immunogen immune animal;
step 3.1 New Zealand white rabbits purchased with SPF grade weighing 2.0KG are divided into an experimental group and a control group, and normal serum is prepared by drawing 0.5ml from the auricular vein of animals before immunization experiment.
Step 3.2, antigen is encapsulated with adjuvant: before each immunization, 400-600ug of the specific antigen-coupled protein complex is sucked into a 2.5ml sterile syringe, the other 2.5ml syringe is used for sucking equivalent complete Freund's adjuvant or Freund's incomplete adjuvant, the two syringes are connected by a sterile plastic hose and repeatedly pumped until the complete emulsification is achieved (one drop is not diffused in cold water, namely, the complete emulsification degree of water-in-oil is achieved), and the specific antigen-coupled protein complex can be used for immunization.
Step 3.3, animal sensitization: 400mg of the specific antigen-coupled protein complex and complete Freund's adjuvant in a volume ratio of 1: step 2, mixing, and injecting the mixture subcutaneously in the trunk, neck and back of the animal by a multipoint method (about 200ul per point) to increase the sensitization effect.
Step 3.4 boost the antigen weekly with the same antigen dose and cross-emulsifying with Freund's incomplete adjuvant and Freund's complete adjuvant, the emulsification method was the same. The injection is respectively carried out at the subcutaneous parts of the back, the subcutaneous parts of the abdomen, the inguinal canal, the popliteal canal, the sole and other parts of the rabbit. During injection, the rabbit is fixed by the rabbit box, the skin is lifted by the left hand, the syringe is held by the right hand, the angle between the needle head and the skin is adjusted to be about 15 degrees, the needle head is injected into the skin for 1-2cm and then lifted upwards to prevent the needle head from penetrating into the muscle, and about 200ul of the needle head is injected at each point.
Step 3.5, specific immunization protocol is shown below, for a total of 10 weeks. Each rabbit was boosted twice by the ear vein at weeks 7 and 9. The Elsia experiments were performed at weeks 4, 8, 9 and 10 to detect antibody titers and specificity, respectively.
Step 4, collecting antibody serum;
and 4.1, fixing four limbs of the rabbit in a supine position by using ropes, wherein the two upper limbs are fixed at the back of the head in a crossed manner, tying the upper jaw and the incisors of the rabbit by using the ropes, and pulling the rabbit backwards to fix the rabbit head on the two upper limbs in a homeopathic manner.
Step 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck along the middle of the neck from the upper fossa sternum to the lower jaw for about 15cm, carefully separating subcutaneous tissues along the trachea after finding out the trachea, and obtaining the mastoid muscle from the distal end to the throat and from the proximal end to the sternoclavicular muscle.
Step 4.3, the beating carotid artery is available below the trachea, the bilateral carotid arteries are carefully isolated and sufficiently dissociated.
Step 4.4, sleeving two black wires into one artery and separating the two black wires (one at the distal end and one at the proximal end), ligating the distal end of the artery by using the wires, clamping the proximal end of the artery by using an arterial clamp, cutting a small opening on the artery wall between the wires and the arterial clamp by using ophthalmic scissors, rapidly inserting a prefabricated thin plastic hose, and rapidly fixing the hose and the artery by using the proximal wire so as to prevent the hose from falling out and leaking blood.
Step 4.5, lightly loosening the arterial clamp, obliquely placing the arterial clamp by using a 50ml centrifuge tube, and increasing the blood discharge amount by treating the carotid artery at the other side in the same way until no blood drips out, and extruding the heart when the blood discharge amount is slow so as to increase the blood discharge amount.
Step 4.6, isolation and preservation of rabbit antisera rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first aspiration of serum, the serum was aspirated a second time by centrifugation at 12000 rpm at 4 degrees celsius for 15 minutes. Finally, about 50ml of SPACA1 antibody serum is obtained, and after being split-packed by a 15ml centrifuge tube, naN3 with the volume of 0.01% is added respectively, and the mixture is placed in a refrigerator with the temperature of minus 20 ℃ for storage.
Step 5, separating and purifying the anti-human sperm membrane protein SPACA1IgG antibody by a saturated ammonium sulfate method;
step 5.1, 3ml of rabbit antiserum was transferred into a 15ml centrifuge tube, 3ml of 0.01M pH7.4PBS was gradually added at 4℃and 6ml of saturated ammonium sulfate solution at pH7.0 was gradually added dropwise with slow shaking.
Step 5.2 when the ammonium sulphate solution reached 50% saturation, the solution was left at 4℃overnight.
Step 5.3, centrifuging the solution at 4 ℃ for 20min at 10000 revolutions per minute, and removing the supernatant to obtain the globulin precipitate.
Step 5.4, after dissolving the precipitate in 3ml of 0.01M PH7.4PBS at 4 ℃, 1.5ml of saturated ammonium sulfate solution (33% saturation of the ammonium sulfate solution) was added dropwise, and the precipitate was taken for 30min. The procedure was repeated twice.
Step 5.5, centrifuging at 4 ℃ for 10min at 10000 revolutions per minute, and discarding the supernatant to obtain antibody IgG precipitate.
Step 5.6, the supernatant was discarded, 3ml of 0.01M PH7.4PBS was added to dissolve the antibody IgG pellet, and the pellet was filled into dialysis bags.
Step 5.7, dialyzing the dialysis bag in 0.01M PH7.4PBS solution at 4deg.C. The liquid is changed for several times until the external liquid for dialysis has no yellow change. 8) And taking a sample in the dialysis bag, drying in vacuum, taking a small amount of sample, diluting by a proper multiple, and measuring the protein content.
Step 6, identifying the binding titer of the serum antibody and the natural human SPACA1 protein by an ELISA;
step 6.1, preparing the human sperm membrane protein soluble antigen.
After 3-5d of forbidden appetite of healthy men, the masturbation method takes semen. After the sperm is liquefied in a water bath at 37 ℃, carrying out routine semen analysis, and collecting semen with the sperm survival rate of more than 60 percent, the sperm motility a grade of more than 25 percent, or the sperm with the sperm motility a grade of more than 50 percent for standby; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form two layers of Percoll density gradient separation columns; 4ml of semen to be used was carefully added to the centrifuge tube and a clear interface was seen. 3000 rpm, centrifuging for 15min, carefully sucking out sperm layer, adding into 2ml PBS, centrifuging at 4deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Clean sperm cell pellet was obtained and thoroughly mixed with about 3-5ml PBS and transferred to a 15ml sterile centrifuge tube to obtain sperm cell suspension.
Step 6.1.2, breaking up sperm cell membranes by using an ultrasonic cytoclasis instrument, and fully exposing antigenic sites by membrane proteins. The method comprises the following steps: 3-5ml of a 15ml centrifuge tube containing sperm cells was placed in crushed ice, the top cap was opened, an ultrasonic cytobreaker probe was placed in the solution, the intensity was adjusted to 70%, and the breaking time was 6min (breaking 5s, suspending 5s to prevent temperature superantigen denaturation and foam generation).
Step 6.1.3, centrifuging the solution (4 ℃ C., 12000 r/min), discarding the precipitate, collecting supernatant (sperm-soluble membrane protein antigen), and preserving at 4 ℃ C. Repeating the steps, collecting the soluble antigens obtained in the steps and storing the soluble antigens in a refrigerator at the temperature of-80 ℃.
Step 6.2, coating, namely diluting the humanized sperm membrane protein antigen to 50ug/ml, dripping the diluted humanized sperm membrane protein antigen into a plurality of small holes of a 96-well plate, 100 mu L/hole and standing at 4 ℃ overnight.
Step 6.3, sealing: the soluble antigen in the wells was discarded, and 150. Mu.L/well of 5% nonfat dry milk (blocking solution) was added and incubated at 37℃for 40min.
Step 6.4, washing, namely adding 200 ul/hole of washing liquid (PBST), washing three times, and standing for 3min each time.
Step 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of serum antibodies to be tested with different dilutions, and standing at 37 ℃ for 40min.
And 6.6, washing, namely adding PBST into the small holes, standing for 3min, discarding the washing liquid in the holes, repeating for three times, and finally fully throwing away the washing liquid in the holes.
Step 6.7, adding enzyme-labeled antibody (secondary antibody) by diluting HRP-labeled goat anti-rabbit secondary antibody by 1:10000, adding 100 μl/well, and standing at 37deg.C for 40min.
Step 6.8, wash with PBST three times, 3min each time.
And 6.9, developing, namely adding 100 mu L/hole of TMB developing solution, and standing for 15min at normal temperature in a dark place.
Step 6.10, stop solution, 1M H2SO4, 50. Mu.L/well.
Step 6.11, measuring, namely measuring the OD 490nm value by using an enzyme-labeled instrument.
Step 7, cell immunofluorescence staining to locate SPACA1 protein distribution on the surface of the sperm;
step 7.1, collection of human sperm and neutrophils: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10minSperm cells were collected and centrifuged 3 times with 0.01mph7.4PBS wash; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 7.2, sperm smear: uniformly dripping 0.5ml PBS solution containing 5×106 sperms on a polylysine coated glass slide, and naturally air-drying at room temperature to form a sperm smear;
step 7.3, wash 3 times with 0.01mph7.4PBS for 5 minutes each;
step 7.4, covering the mixture with 4% paraformaldehyde at room temperature for fixing for 15 minutes, and avoiding light;
step 7.5, washing 3 times by using 0.01MPH7.4PB PBS buffer solution for 5 minutes each time;
step 7.6, covering cells with 0.25% Triton X-100 (permeant) for 5-7 min (note: this step can be omitted without permeant);
step 7.7, 0.01MPH7.4PBS three washes of 5 minutes each;
step 7.8, goat sealing serum is sealed for 40 minutes at room temperature;
step 7.9, preparing a primary antibody, diluting SPACA1 to 150ug/ml, adding the primary antibody, and keeping the wet box at 4 ℃ overnight in a dark place;
step 7.10, washing with PBS three times, each for 5 minutes;
step 7.11, preparing fluorescence labeled secondary antibody (Ab 150079) coat anti-rabit IgG (H & L), diluting with PBS according to a ratio of 1:100, and preserving in a dark place;
step 7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (in the dark);
step 7.13, washing three times with PBS at 4 ℃ for 15 minutes (light shielding);
step 7.14, the DAPI is used for dying the nucleus, and the cells are completely covered (light shielding);
in step 7.15, the stained cells were observed with a confocal microscope, and cells exhibiting high-intensity red fluorescence (emission wavelength of 647 nm) were observed and photographed as an observation field.
Step 8, incubating normal mature human sperm with SPACA1 antibody;
step 8.1, collection of normal mature sperm and neutrophils of human origin: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate, and adjusting the concentration of sperm and neutrophil to 2×106/ml;
step 8.2, co-incubation of antibody and sperm: uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of SPACA1 antibody solution of 2mg/ml respectively, incubating at 37 ℃ for 30 minutes, washing with 0.01MPH7.4PBS, centrifuging for 3 times, and fixing volume to 10ml; the subsequent steps are the same as steps 7.2 to 7.15.
The invention has the advantages and beneficial effects that:
the anti-SPACA 1 antibody prepared by the invention not only has stable specific binding capacity with immobilized and denatured SPACA1 protein antigen site, but also can specifically bind with the SPACA1 protein antigen site of the sperm acrosome membrane which keeps the integrity of cells in physiological state, thus obtaining specific antibody orientation effect. The antibody can construct a composite targeting system by linking complex multicomponent and multifunctional biological groups at the Fc end according to specific application conditions so as to meet different requirements. The SPACA1 protein is widely distributed on the outer membrane of the acrosome region of the sperm, and the antibody prepared by the invention has good application prospect in the fields of targeting tracing of the sperm, biological pharmacy, accurate treatment and the like.
Drawings
FIG. 1 amino acid sequence of human SPACA1 and amino acid sequence of specific SPACA1 epitope.
FIG. 2TMHMM2.0 predicts that the transmembrane region of SPACA1 protein is at positions 217-239 and amino acids 1-216 are outside the cell membrane.
Fig. 3s ignalp 4.1 predicts that SPACA1 is a secreted protein with sequence cleavage site at position 30 smax=0.967 and ymax=0.840.
FIG. 4ExPASY-ProtScale predicts that SPACA1 protein sequence has 6 forward peaks, amino acid at position 238 (ILE, isoleucine) has the highest score, indicating that the amino acid at this position is most hydrophobic; 5 distinct negative peaks, amino acid at position 53 (Glu ) has the lowest score, indicating that the amino acid at this position is most hydrophilic.
FIG. 5ELSIA assay of SPACA1 antibody titers, (A) fourth week antibody titers of 1:1600; (B) the eighth week antibody titer is 1:1600; (C) the ninth week antibody titer is 1:3200; (D) The titer at week ten was 1:3200, with the highest OD for the antibodies at week ten.
FIG. 6 as a negative control, anti-human IgG antibodies were not stained on human sperm.
FIG. 7SPACA1 protein was concentrated mainly in the acrosome region of the sperm head, with no staining seen in neutrophils.
FIG. 8 shows that SPACA1 protein is in the in vivo state of sperm, and the epitope is located in the acrocoel of sperm and cannot be directly combined with the antibody outside the sperm cell membrane.
Detailed Description
Example 1:
the preparation method of the human sperm membrane protein SPACA1 specific antigen coupling protein and the antibody specifically comprises the following steps:
step 1, analyzing and designing specific antigen epitope of human sperm membrane protein SPACA 1;
the amino acid sequence of SPACA1 protein is subjected to antigen epitope analysis by using biological information software, indexes such as transmembrane fragments, polypeptide activity, hydrophilicity, antigenicity and the like are mainly evaluated, and the practical experience of preparing antibodies in the past is combined, so that 15 amino acids at 61-74 positions of the SPACA1 protein are finally determined, a cysteine is modified at the C end of the polypeptide, and the amino acid sequence of the synthesized polypeptide is NYAPPETEDVSNRNC (figure 1).
Step 1.1, online predicting a transmembrane region of a humanized sperm membrane protein SPACA1 by using TMHMM;
logging in a TMHMM main page, submitting a FASTA format SPACA1 protein sequence in a popup box of a 'select file' or pasting the sequence in a text box under 'OR by pasting sequence(s) in FASTA format'; out formats have three options, respectively, graphical output (existence); text output (no graphics); displaying proteins line by line (One line protein), and displaying a default option graphically; after the adjustment is finished, submitting and checking a prediction analysis result by pressing a submit key, wherein the transmembrane region of the SPACA1 protein sequence is positioned at 217-239, and the 1-216 is positioned in the inner membrane of the top body region; (FIG. 2).
Step 1.2, predictive analysis of signal peptide is carried out on human sperm membrane protein SPACA1 by using SignalP 4.1 Server;
logging in a SignalP 4.1 Server main page, submitting a FASTA format protein sequence in a pop-up box of a 'selected file' or pasting the sequence in a text box below; parameter setting organosm group: selecting Eukaryotes; d-cutoff values: selecting Default (optimized for correlation); graphics output: selecting PNG (inline); output format: a Standard; method selection Input sequences may include TM regions. Submitting and checking a prediction analysis result by pressing a submit key after parameter setting is finished, wherein the cleavage site of the SPACA1 protein sequence is at 30 positions, smax=0.967 and Ymax=0.840; (FIG. 3).
Step 1.3, predicting and analyzing hydrophobicity of the humanized 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 accession number (AC)'; or the FASTA format protein sequence is stuck to a text box of Or you can paste your own sequence in the box below; the default setting is selected, the predicted analysis result is submitted and checked according to the 'sub' after the setting is finished, the SPACA1 protein sequence has 6 forward peaks, and the 238 th amino acid (ILE, isoleucine) has the highest score, which indicates that the amino acid of the site has the strongest hydrophobicity. The 5 distinct negative peaks, amino acid at position 53 (Glu ) has the lowest score, indicating that the amino acid at this position is most hydrophilic (FIG. 4).
Step 2, preparing and purifying the human sperm membrane protein SPACA1 specific antigen coupling protein;
step 2.1, synthesizing SPACA1 artificial polypeptide;
the Shanghai Hua big gene company adopts a polypeptide automatic synthesizer to synthesize and purify SPACA1 (16 peptide) by 10mg, and adopts mass spectrometry and high performance liquid chromatography to carry out purity identification, wherein the purity is more than 95 percent.
Step 2.2, combining an EDC couple with an adult sperm membrane protein SPACA1 specific antigen coupling protein;
KLH was prepared as a 10mg/ml solution of 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 of EDC solution 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 of reaction at room temperature.
Step 2.3, purifying the human sperm membrane protein SPACA1 specific antigen coupling protein complex;
60ml of ultra-pure water degassed by ultrasonic vibration is added into a Purification Buffer Salts bottle to dissolve the content, a desalting column is used for filtering every 0.5ml of sample, 0.5ml of human sperm membrane protein SPACA1 specific antigen coupling protein complex is gently dripped into 25ml of Purification Buffer Salts eluting column, standing is carried out for 2 minutes, 5ml of purification buffer is taken for eluting the column, the eluent is collected, absorbance is detected at 280nm by ultraviolet-visible absorption spectrometry, and the content of the polypeptide-KLH complex is calculated according to the absorption peak. If the immunogen needs to be stored for a longer period of time, the immunogen can be filtered using a 0.22um filter and stored at-20 ℃.
Step 3, preparing an artificial immunogen immune animal;
step 3.1, purchase 4 New Zealand white rabbits with SPF grade and weight of 2.0 KG. The normal serum was prepared from 0.5ml of animal ear vein blood drawn prior to immunization experiments, divided into SPACA1 group and control group.
Step 3.2, antigen is encapsulated with adjuvant: before each immunization, 400-600ug of the human sperm membrane protein SPACA1 specific antigen coupling protein complex is inhaled into a 2.5ml sterile syringe, the other 2.5ml syringe is used for inhaling the equivalent amount of complete Freund adjuvant or Freund incomplete adjuvant, and the two syringes are connected by a sterile plastic hose and repeatedly pumped until the mixture is completely emulsified (one drop is not diffused in cold water, namely, the complete emulsification degree of water-in-oil is achieved), so that the mixture can be used for immunization.
Step 3.3, animal sensitization: 400mg of human sperm membrane protein SPACA1 specific antigen coupled protein complex and complete Freund's adjuvant according to the volume ratio of 1: step 2, mixing, and injecting the mixture subcutaneously in the trunk, neck and back of the animal by a multipoint method (about 200ul per point) to increase the sensitization effect.
Step 3.4 boost the antigen weekly with the same antigen dose and cross-emulsifying with Freund's incomplete adjuvant and Freund's complete adjuvant, the emulsification method was the same. The injection is respectively carried out at the subcutaneous parts of the back, the subcutaneous parts of the abdomen, the inguinal canal, the popliteal canal, the sole and other parts of the rabbit. During injection, the rabbit is fixed by the rabbit box, the skin is lifted by the left hand, the syringe is held by the right hand, the angle between the needle head and the skin is adjusted to be about 15 degrees, the needle head is injected into the skin for 1-2cm and then lifted upwards to prevent the needle head from penetrating into the muscle, and about 200ul of the needle head is injected at each point. 5-10ml of blood was collected as immune serum from rabbit ear vein after each immunization.
Step 3.5, specific immunization protocol is shown below, for a total of 10 weeks. Each rabbit was boosted twice by the ear vein at weeks 7 and 9. The Elsia experiments were performed at weeks 4, 8, 9 and 10 to detect antibody titers and specificity, respectively.
Step 4, preparing antibody serum;
and 4.1, fixing four limbs of the rabbit in a supine position by using ropes, wherein the two upper limbs are fixed at the back of the head in a crossed manner, tying the upper jaw and the incisors of the rabbit by using the ropes, and pulling the rabbit backwards to fix the rabbit head on the two upper limbs in a homeopathic manner.
Step 4.2, exposing the neck, cutting off the hair of the neck after disinfection, cutting off the skin of the neck along the middle of the neck from the upper fossa sternum to the lower jaw for about 15cm, carefully separating subcutaneous tissues along the trachea after finding out the trachea, and obtaining the mastoid muscle from the distal end to the throat and from the proximal end to the sternoclavicular muscle.
Step 4.3, the beating carotid artery is available below the trachea, the bilateral carotid arteries are carefully isolated and sufficiently dissociated.
Step 4.4, sleeving two black wires into one artery and separating the two black wires (one at the distal end and one at the proximal end), ligating the distal end of the artery by using the wires, clamping the proximal end of the artery by using an arterial clamp, cutting a small opening on the artery wall between the wires and the arterial clamp by using ophthalmic scissors, rapidly inserting a prefabricated thin plastic hose, and rapidly fixing the hose and the artery by using the proximal wire so as to prevent the hose from falling out and leaking blood.
Step 4.5, lightly loosening the arterial clamp, obliquely placing the arterial clamp by using a 50ml centrifuge tube, and increasing the blood discharge amount by treating the carotid artery at the other side in the same way until no blood drips out, and extruding the heart when the blood discharge amount is slow so as to increase the blood discharge amount. 70ml of SPACA1 rabbit blood was obtained by this method.
Step 4.6, isolation and preservation of rabbit antisera rabbit serum was placed in a refrigerator at 4 ℃ overnight. After the first aspiration of serum, the serum was aspirated a second time by centrifugation at 12000 rpm at 4 degrees celsius for 15 minutes. Finally, about 50ml of SPACA1 antibody serum is obtained, and after being split-packed by a 15ml centrifuge tube, naN3 with the volume of 0.01% is added respectively, and the mixture is placed in a refrigerator with the temperature of minus 20 ℃ for storage.
Step 5, purifying the antibody by a saturated ammonium sulfate method;
step 5.1, 3ml of rabbit antiserum was transferred into a 15ml centrifuge tube, 3ml of 0.01M pH7.4PBS was gradually added at 4℃and 6ml of saturated ammonium sulfate solution at pH7.0 was gradually added dropwise with slow shaking.
Step 5.2 when the ammonium sulphate solution reached 50% saturation, the solution was left at 4℃overnight.
Step 5.3, centrifuging the solution at 4 ℃ for 20min at 10000 revolutions per minute, and removing the supernatant to obtain the globulin precipitate.
Step 5.4, after dissolving the precipitate in 3ml of 0.01M PH7.4PBS at 4 ℃, 1.5ml of saturated ammonium sulfate solution (33% saturation of the ammonium sulfate solution) was added dropwise, and the precipitate was taken for 30min. The procedure was repeated twice.
Step 5.5, centrifuging at 4 ℃ for 10min at 10000 revolutions per minute, and discarding the supernatant to obtain antibody IgG precipitate.
Step 5.6, the supernatant was discarded, 3ml of 0.01M PH7.4PBS was added to dissolve the antibody IgG pellet, and the pellet was filled into dialysis bags.
Step 5.7, dialyzing the dialysis bag in 0.01M PH7.4PBS solution at 4deg.C. The liquid is changed for several times until the external liquid for dialysis has no yellow change. 8) And taking a sample in the dialysis bag, drying in vacuum, taking a small amount of sample, diluting by a proper multiple, and measuring the protein content.
Step 6, identifying the binding titer of the serum SPACA1 antibody and the non-denatured human SPACA1 protein by an ELISA;
step 6.1, preparing a human sperm membrane protein soluble antigen;
after 3-5d of forbidden appetite of healthy men, the masturbation method takes semen. After the sperm is liquefied in a water bath at 37 ℃, carrying out routine semen analysis, and collecting semen with the sperm survival rate of more than 60 percent, the sperm motility a grade of more than 25 percent, or the sperm with the sperm motility a grade of more than 50 percent for standby; sequentially adding 5ml of 90% and 45% Percoll liquid into a 15ml centrifuge tube to form two layers of Percoll density gradient separation columns; 4ml of semen to be used was carefully added to the centrifuge tube and a clear interface was seen. 3000 rpm, centrifuging for 15min, carefully sucking out sperm layer, adding into 2ml PBS, centrifuging at 4deg.C for 10min at 3000 rpm to obtain sperm cell precipitate, mixing with 4deg.C sterile PBS, centrifuging under the above conditions, and repeating for three times. Clean sperm cell pellet was obtained and thoroughly mixed with about 3-5ml PBS and transferred to a 15ml sterile centrifuge tube to obtain sperm cell suspension.
Step 6.1.2, breaking up sperm cell membranes by using an ultrasonic cytoclasis instrument, and fully exposing antigenic sites by membrane proteins. The method comprises the following steps: 3-5ml of a 15ml centrifuge tube containing sperm cells was placed in crushed ice, the top cap was opened, an ultrasonic cytobreaker probe was placed in the solution, the intensity was adjusted to 70%, and the breaking time was 6min (breaking 5s, suspending 5s to prevent temperature superantigen denaturation and foam generation).
Step 6.1.3, centrifuging the solution (4 ℃ C., 12000 r/min), discarding the precipitate, collecting supernatant (sperm-soluble membrane protein antigen), and preserving at 4 ℃ C. Repeating the steps, collecting the soluble antigens obtained in the steps and storing the soluble antigens in a refrigerator at the temperature of-80 ℃.
Step 6.2, coating, namely diluting the humanized sperm membrane protein antigen to 50ug/ml, dripping the diluted humanized sperm membrane protein antigen into a plurality of small holes of a 96-well plate, 100 mu L/hole and standing at 4 ℃ overnight.
Step 6.3, sealing: the soluble antigen in the wells was discarded, and 150. Mu.L/well of 5% nonfat dry milk (blocking solution) was added and incubated at 37℃for 40min.
Step 6.4, washing, namely adding 200 ul/hole of washing liquid (PBST), washing three times, and standing for 3min each time.
Step 6.5, adding a primary antibody, namely fully throwing away the washing liquid in the hole, sequentially adding 100 mu L/hole of serum antibodies to be tested with different dilutions, and standing at 37 ℃ for 40min.
And 6.6, washing, namely adding PBST into the small holes, standing for 3min, discarding the washing liquid in the holes, repeating for three times, and finally fully throwing away the washing liquid in the holes.
Step 6.7, adding enzyme-labeled antibody (secondary antibody) by diluting HRP-labeled goat anti-rabbit secondary antibody by 1:10000, adding 100 μl/well, and standing at 37deg.C for 40min.
Step 6.8, wash with PBST three times, 3min each time.
And 6.9, developing, namely adding 100 mu L/hole of TMB developing solution, and standing for 15min at normal temperature in a dark place.
Step 6.10, stop solution, 1M H2SO4, 50. Mu.L/well.
Step 6.11, measurement: OD 490nm was measured using a microplate reader. After ten immunizations in this experiment, the titers of the final SPACA1 antibodies at weeks four, eight, ninth and tenth were 1:1600, 1:3200 and 1:3200, respectively, with the ten week antibody OD values being highest (FIG. 5).
Step 7, cell immunofluorescence staining to locate SPACA1 protein distribution on the surface of the sperm;
step 7.1, collection of human sperm and neutrophils: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Counting with a blood cell counting plate to adjust the concentration of sperm and neutrophil to 2×10 6 Individual/ml;
step 7.2, sperm smear: uniformly dripping 0.5ml PBS solution containing 5×106 sperms on a polylysine coated glass slide, and naturally air-drying at room temperature to form a sperm smear;
step 7.3, wash 3 times with 0.01mph7.4PBS for 5 minutes each;
step 7.4, covering the mixture with 4% paraformaldehyde at room temperature for fixing for 15 minutes, and avoiding light;
step 7.5, washing 3 times by using 0.01MPH7.4PB PBS buffer solution for 5 minutes each time;
step 7.6, covering cells with 0.25% Triton X-100 (permeant) for 5-7 min (note: this step can be omitted without permeant);
step 7.7, 0.01MPH7.4PBS three washes of 5 minutes each;
step 7.8, goat sealing serum is sealed for 40 minutes at room temperature;
step 7.9, preparing a primary antibody, diluting SPACA1 to 150ug/ml, adding the primary antibody, and keeping the wet box at 4 ℃ overnight in a dark place;
step 7.10, washing with PBS three times, each for 5 minutes;
step 7.11, preparing fluorescence labeled secondary antibody (Ab 150079) coat anti-rabit IgG (H & L), diluting with PBS according to a ratio of 1:100, and preserving in a dark place; .
Step 7.12, adding a secondary antibody, and incubating for 1 hour at room temperature (in the dark);
step 7.13, washing three times with PBS at 4 ℃ for 15 minutes (light shielding);
step 7.14, the DAPI is used for dying the nucleus, and the cells are completely covered (light shielding);
in step 7.15, the stained cells were observed with a confocal microscope, and cells exhibiting high-intensity red fluorescence (emission wavelength of 647 nm) were observed and photographed as an observation field. Immunofluorescent staining of human sperm and neutrophil smears showed that IgG antibodies were not stained on human sperm (fig. 6); the SPACA1 protein was mainly concentrated in the acrosome region of the sperm head and no staining was seen in neutrophils (FIG. 7).
Step 8, incubating normal mature human sperm with SPACA1 antibody;
step 8.1, collection of normal mature sperm and neutrophils of human origin: collecting semen of healthy person by masturbation method, liquefying completely at 37deg.C, centrifuging at 2000 rpm for 10min, collecting sperm cells, washing with 0.01MPH7.4PBS, and centrifuging for 3 times; human neutrophils were collected by gradient centrifugation and lysed by dilution with the same volume of 0.01 mM H7.4 PBS. Using blood cell counting plates in combinationCounting, and regulating sperm and neutrophil concentration to 2×10 6 Individual/ml;
step 8.2, co-incubation of antibody and sperm: uniformly mixing 0.5ml of sperm solution or neutrophil solution with 0.5ml of SPACA1 antibody solution of 2mg/ml respectively, incubating at 37 ℃ for 30 minutes, washing with 0.01MPH7.4PBS, centrifuging for 3 times, and fixing volume to 10ml; the subsequent operation steps are the same as the 7.2 th to 7.15 th steps; the co-incubation experiment of the SPACA1 antibody and the sperm can observe the expression condition of the antibody combined with the motile sperm SPACA1 protein, and the co-incubation experiment shows that the SPACA1 protein is in the living state of the sperm, and the epitope is positioned in the acrocoel of the sperm and can not be directly combined with the antibody outside the sperm cell membrane. (FIG. 8).
Claims (7)
1. The N-terminal human sperm membrane protein SPACA1 specific polypeptide is characterized in that an antigen binding site is positioned on an outer membrane of a human sperm acrosome region, a polypeptide sequence is a peptide segment from 61 st to 74 th amino acids in an amino acid sequence of the human sperm membrane protein SPACA1, cysteine is added at a C terminal, and the amino acid sequence of the polypeptide is as follows: NYAPPETEDVSNRNC.
2. A human sperm membrane protein SPACA1 specific antigen coupling protein is characterized in that a mixture of polypeptide amino acid hydrophobic groups and keyhole limpet hemocyanin amino groups are covalently crosslinked through a crosslinking agent for synthesizing antigen polypeptide NYAPPETEDVSNRNC.
3. A method for preparing an antibody against human sperm membrane protein SPACA1, which is characterized by comprising the following steps:
1) The analysis and design of specific antigen epitope of human sperm membrane protein SPACA 1;
carrying out antigen epitope analysis on the amino acid sequence of the protein by utilizing biological information software, and mainly evaluating the activity, hydrophilicity and antigenicity index of the transmembrane segment and the polypeptide; the determined extracellular specificity antigen epitope sequence is a peptide segment of 61 th to 74 th amino acids in the amino acid sequence of human sperm membrane protein SPACA1, cysteine is added at the C end, and the amino acid sequence of the polypeptide is as follows: NYAPPETEDVSNRNC;
2) Preparing and purifying human sperm membrane protein SPACA1 specific antigen coupling protein;
3) Preparing an artificial immunogen immune animal;
4) Collecting antibody serum;
5) Separating and purifying an anti-human sperm membrane protein SPACA1IgG antibody by a saturated ammonium sulfate method;
6) The ELISA experiments are used for identifying the binding titer of the serum antibody and the natural human SPACA1 protein;
7) Cell immunofluorescence staining locates SPACA1 protein distribution on sperm surface.
4. The method of claim 3, wherein the conjugated protein is keyhole limpet hemocyanin.
5. The method of claim 3, wherein the artificial immunogen is a human sperm membrane protein SPACA1 specific antigen coupled protein of claim 2 mixed with complete or incomplete Freund's adjuvant.
6. The method of claim 3, wherein the immunized animal is a human sperm membrane protein extract having a serum titer greater than 1 after multiple subcutaneous injections in the back of the animal and two or more booster immunizations: 3200.
7. the method for producing an antibody according to claim 3, wherein the isolated and purified anti-human sperm membrane protein SPACA1IgG antibody is an IgG antibody which can be obtained in high purity from antibody serum by ammonium persulfate 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 CN114395028A (en) | 2022-04-26 |
| CN114395028B true 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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010129310A1 (en) * | 2009-04-27 | 2010-11-11 | Roswell Park Cancer Institute | Reagents and methods for producing bioactive secreted peptides |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU784015B2 (en) * | 2000-01-19 | 2006-01-12 | University Of Virginia Patent Foundation | Sperm specific proteins |
| JP6108338B2 (en) * | 2009-06-25 | 2017-04-05 | 国立大学法人 千葉大学 | Methods for identifying compounds that inhibit fertilization |
-
2022
- 2022-02-09 CN CN202210120812.4A patent/CN114395028B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010129310A1 (en) * | 2009-04-27 | 2010-11-11 | Roswell Park Cancer Institute | Reagents and methods for producing bioactive secreted peptides |
| 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 |
|---|
| sperm acrosome membrane-associated protein 1 precursor [Homo sapiens];Zhang T等;GenBanK;第3-4页CDS和ORIGIN部分 * |
| 精子表面透明带蛋白受体的分析与功能研究;李秋华;周泽荣;祝晓丽;谭颖;钟小英;姜荣华;宋革;;实用医院临床杂志(第01期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114395028A (en) | 2022-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Telfer | Immunological studies of insect metamorphosis: II. The role of a sex-limited blood protein in egg formation by the Cecropia silkworm | |
| CN110272490B (en) | Targeted CTLA-4 antibody, preparation method and application thereof | |
| CN113896769B (en) | Preparation method of human sperm membrane protein SPAG8 specific polypeptide and antibody | |
| WO2022166806A1 (en) | Novel cd271-based antigenic epitope and application thereof | |
| CN112979817B (en) | Monoclonal antibody for recognizing anti-CLDN 18_2 antibody and preparation method and application thereof | |
| CN114395028B (en) | Preparation method of human sperm membrane protein SPACA1 specific antigen coupling protein and antibody | |
| EP0142345A2 (en) | Anti-idiotypic vaccine employing anti-idiotypic monoclonal antibodies | |
| CN114133442B (en) | Preparation method of human sperm membrane protein SPACA3 specific polypeptide and antibody | |
| CN109082412A (en) | A kind of monoclonal antibody of studies on rhoptry proteins of Toxoplasma gondii ROP18, the cell strain for secreting the antibody and application thereof | |
| CN101906163B (en) | Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof | |
| CN117074674B (en) | Colloidal gold test paper for detecting N-acetyl-beta-D glucosaminidase and preparation method thereof | |
| CN114349846B (en) | Preparation method of human sperm membrane protein OR1D2 specific polypeptide and antibody | |
| CN107298697A (en) | Human PD-L1 protein Y123Site phosphorylation antibody and preparation method and application thereof | |
| Srinivasan et al. | Induction of autoantibodies against tyrosinase-related proteins following DNA vaccination: unexpected reactivity to a protein paralogue | |
| CN115290895A (en) | Application of methylation modification of 162 th lysine of human PD-L1 protein in prediction of immunotherapy sensitivity of malignant tumor | |
| CZ20013082A3 (en) | Peptide | |
| Gao et al. | Autoantibody against cardiac β1-adrenoceptor induces apoptosis in cultured neonatal rat cardiomyocytes | |
| EP0582450A2 (en) | Anti-oxytocin receptor antibodies and methods for their production | |
| CN101602794A (en) | A kind of human MGF polypeptide and preparation method for antibody thereof | |
| CN101921336A (en) | Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof | |
| CN108794631A (en) | A kind of preparation method of the anti-mouse CD36 monoclonal antibodies of mouse | |
| EP0653440B1 (en) | Monoclonal antibodies against hair follicle, hybridomas producing them | |
| CN113512106B (en) | Synthetic peptide of grass carp C5aR protein and polyclonal antibody thereof | |
| CN106478802B (en) | TNF-α protein B cell epitope, multiple antigenic peptide and application containing this epitope | |
| CN116514980A (en) | anti-SIRP alpha antibody and application thereof |
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 |