CN117586406A - 类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 - Google Patents
类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 Download PDFInfo
- Publication number
- CN117586406A CN117586406A CN202311664592.2A CN202311664592A CN117586406A CN 117586406 A CN117586406 A CN 117586406A CN 202311664592 A CN202311664592 A CN 202311664592A CN 117586406 A CN117586406 A CN 117586406A
- Authority
- CN
- China
- Prior art keywords
- pkcs
- dna
- protein
- cancer
- polyclonal antibody
- 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.)
- Pending
Links
- 102100022204 DNA-dependent protein kinase catalytic subunit Human genes 0.000 title claims abstract description 59
- 101710157074 DNA-dependent protein kinase catalytic subunit Proteins 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 102000044159 Ubiquitin Human genes 0.000 title abstract description 9
- 108090000848 Ubiquitin Proteins 0.000 title abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 claims abstract description 32
- 239000000427 antigen Substances 0.000 claims abstract description 21
- 102000036639 antigens Human genes 0.000 claims abstract description 21
- 108091007433 antigens Proteins 0.000 claims abstract description 21
- 241001465754 Metazoa Species 0.000 claims abstract description 10
- 230000003053 immunization Effects 0.000 claims abstract description 7
- 230000034512 ubiquitination Effects 0.000 claims abstract description 7
- 238000010798 ubiquitination Methods 0.000 claims abstract description 7
- 206010028980 Neoplasm Diseases 0.000 claims description 28
- 238000001514 detection method Methods 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 210000002966 serum Anatomy 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 12
- 102100031911 NEDD8 Human genes 0.000 claims description 8
- 108700004934 NEDD8 Proteins 0.000 claims description 7
- 101150107958 NEDD8 gene Proteins 0.000 claims description 7
- 101100532088 Oryza sativa subsp. japonica RUB2 gene Proteins 0.000 claims description 6
- 101100532090 Oryza sativa subsp. japonica RUB3 gene Proteins 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 101150024074 rub1 gene Proteins 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 3
- 208000026310 Breast neoplasm Diseases 0.000 claims description 3
- 206010009944 Colon cancer Diseases 0.000 claims description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- -1 ulml Proteins 0.000 claims description 3
- 108010061408 Autophagy-Related Protein 12 Proteins 0.000 claims description 2
- 102000012035 Autophagy-Related Protein 12 Human genes 0.000 claims description 2
- 108010032769 Autophagy-Related Protein 8 Family Proteins 0.000 claims description 2
- 102000007353 Autophagy-Related Protein 8 Family Human genes 0.000 claims description 2
- 206010005003 Bladder cancer Diseases 0.000 claims description 2
- 206010014733 Endometrial cancer Diseases 0.000 claims description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 2
- 102100034003 FAU ubiquitin-like and ribosomal protein S30 Human genes 0.000 claims description 2
- 101000732045 Homo sapiens FAU ubiquitin-like and ribosomal protein S30 Proteins 0.000 claims description 2
- 101000824318 Homo sapiens Protocadherin Fat 1 Proteins 0.000 claims description 2
- 101000662056 Homo sapiens Ubiquitin D Proteins 0.000 claims description 2
- 101000643925 Homo sapiens Ubiquitin-fold modifier 1 Proteins 0.000 claims description 2
- 101000772767 Homo sapiens Ubiquitin-like protein 5 Proteins 0.000 claims description 2
- 101001057508 Homo sapiens Ubiquitin-like protein ISG15 Proteins 0.000 claims description 2
- 101100436336 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) apg-12 gene Proteins 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 102100022095 Protocadherin Fat 1 Human genes 0.000 claims description 2
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 2
- 102100037932 Ubiquitin D Human genes 0.000 claims description 2
- 102100021012 Ubiquitin-fold modifier 1 Human genes 0.000 claims description 2
- 102100030580 Ubiquitin-like protein 5 Human genes 0.000 claims description 2
- 102100027266 Ubiquitin-like protein ISG15 Human genes 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 201000010536 head and neck cancer Diseases 0.000 claims description 2
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 2
- 208000008732 thymoma Diseases 0.000 claims description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 2
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 108010005705 Ubiquitinated Proteins Proteins 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 12
- 238000012986 modification Methods 0.000 abstract description 12
- 238000002649 immunization Methods 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 17
- 230000000694 effects Effects 0.000 description 16
- 238000002965 ELISA Methods 0.000 description 14
- 230000005782 double-strand break Effects 0.000 description 13
- 241000283973 Oryctolagus cuniculus Species 0.000 description 11
- 238000011534 incubation Methods 0.000 description 10
- 238000004949 mass spectrometry Methods 0.000 description 10
- 230000026731 phosphorylation Effects 0.000 description 9
- 238000006366 phosphorylation reaction Methods 0.000 description 9
- 230000008439 repair process Effects 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 230000027455 binding Effects 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 230000005778 DNA damage Effects 0.000 description 5
- 231100000277 DNA damage Toxicity 0.000 description 5
- 108091000080 Phosphotransferase Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- 230000006780 non-homologous end joining Effects 0.000 description 5
- 102000020233 phosphotransferase Human genes 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000002033 PVDF binder Substances 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 230000007018 DNA scission Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000009527 neddylation Effects 0.000 description 3
- 238000001959 radiotherapy Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 2
- 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
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- 102100023877 E3 ubiquitin-protein ligase RBX1 Human genes 0.000 description 2
- 101710095156 E3 ubiquitin-protein ligase RBX1 Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102100034533 Histone H2AX Human genes 0.000 description 2
- 101000670537 Homo sapiens E3 ubiquitin-protein ligase RNF168 Proteins 0.000 description 2
- 101001067891 Homo sapiens Histone H2AX Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 101710176384 Peptide 1 Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 101710178916 RING-box protein 1 Proteins 0.000 description 2
- 102000004909 RNF168 Human genes 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102000000504 Tumor Suppressor p53-Binding Protein 1 Human genes 0.000 description 2
- 108010041385 Tumor Suppressor p53-Binding Protein 1 Proteins 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 2
- 102100036973 X-ray repair cross-complementing protein 5 Human genes 0.000 description 2
- 101710124921 X-ray repair cross-complementing protein 5 Proteins 0.000 description 2
- 102100036976 X-ray repair cross-complementing protein 6 Human genes 0.000 description 2
- 101710124907 X-ray repair cross-complementing protein 6 Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000011091 antibody purification Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- TWBYWOBDOCUKOW-UHFFFAOYSA-N isonicotinic acid Chemical compound OC(=O)C1=CC=NC=C1 TWBYWOBDOCUKOW-UHFFFAOYSA-N 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000011127 radiochemotherapy Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- KIAPWMKFHIKQOZ-UHFFFAOYSA-N 2-[[(4-fluorophenyl)-oxomethyl]amino]benzoic acid methyl ester Chemical compound COC(=O)C1=CC=CC=C1NC(=O)C1=CC=C(F)C=C1 KIAPWMKFHIKQOZ-UHFFFAOYSA-N 0.000 description 1
- 101100243447 Arabidopsis thaliana PER53 gene Proteins 0.000 description 1
- 101100220616 Caenorhabditis elegans chk-2 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108010088874 Cullin 1 Proteins 0.000 description 1
- 102100039195 Cullin-1 Human genes 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000005971 DNA damage repair Effects 0.000 description 1
- 102100027828 DNA repair protein XRCC4 Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101100191383 Dictyostelium discoideum dnapkcs gene Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102100030013 Endoribonuclease Human genes 0.000 description 1
- 101710199605 Endoribonuclease Proteins 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 102100029075 Exonuclease 1 Human genes 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 101000649315 Homo sapiens DNA repair protein XRCC4 Proteins 0.000 description 1
- 101001107071 Homo sapiens E3 ubiquitin-protein ligase RNF8 Proteins 0.000 description 1
- 101000918264 Homo sapiens Exonuclease 1 Proteins 0.000 description 1
- 101000578059 Homo sapiens Non-homologous end-joining factor 1 Proteins 0.000 description 1
- 101000628899 Homo sapiens Small ubiquitin-related modifier 1 Proteins 0.000 description 1
- 101000832643 Homo sapiens Small ubiquitin-related modifier 4 Proteins 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100342585 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mus-51 gene Proteins 0.000 description 1
- 102100028156 Non-homologous end-joining factor 1 Human genes 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000004910 RNF8 Human genes 0.000 description 1
- 101100342589 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pku70 gene Proteins 0.000 description 1
- 101710113029 Serine/threonine-protein kinase Proteins 0.000 description 1
- 102100026940 Small ubiquitin-related modifier 1 Human genes 0.000 description 1
- 102100024535 Small ubiquitin-related modifier 4 Human genes 0.000 description 1
- 101150080074 TP53 gene Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102100020695 Ubiquitin-conjugating enzyme E2 N Human genes 0.000 description 1
- 101710192923 Ubiquitin-conjugating enzyme E2 N Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000012820 cell cycle checkpoint Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 201000005621 esophagus lymphoma Diseases 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000022244 formylation Effects 0.000 description 1
- 238000006170 formylation reaction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 101150085005 ku70 gene Proteins 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 150000003906 phosphoinositides Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000028617 response to DNA damage stimulus Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/11—Protein-serine/threonine kinases (2.7.11)
- C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明属于生物工程技术领域，具体的涉及一种泛素化修饰Neddylation的DNA‑PKcs蛋白的多克隆抗体及其制备方法。本发明根据DNA‑PKcs蛋白序列，设计并合成类泛素化抗原多肽，之后用于动物免疫，制备多克隆抗体。
Description
技术领域
本发明属于生物工程技术领域，具体的涉及一种泛素化修饰Neddylation的DNA-PKcs蛋白的多克隆抗体及其制备方法。
背景技术
DNA-PKcs与ATM、ATR同属于PI3KK(phosphoinositide 3-kinase(PI3K)-relatedkinases)家族，是一种丝氨酸/苏氨酸蛋白激酶，三者有相似的结构域和很多共同的结构特征，它们的激酶结构域位于蛋白的C端，在激酶结构域的上游和下游分别是FAT(FRAP-ATM-TRRAP)结构域和FATC(FAT C-terminal)结构域。由于结构的相似性三者有很多共同的底物，功能上有部分重叠，并且在DNA损伤应答过程中都发挥着至关重要的作用。
DNA-PKcs由prkdc基因编码，进化上保守，几乎所有哺乳动物细胞都表达。DNA-PKcs蛋白有4128个氨基酸残基，主要含有一个催化结构域、DNA结合结构域和Ku结合结构域。DNA-PKcs和ATM主要响应DSBs，分别被Ku70/Ku80和MRN复合物募集到DNA断裂末端，ATR则主要响应DNA复制压力，通过与ATRIP的互作募集到DNA断裂末端。早期生化研究发现DNA-PK在磷酸化其底物蛋白时更加偏爱后面是谷氨酰胺的丝氨酸或苏氨酸(S/T-Q)，后来发现ATM和ATR也是如此。
DNA双链断裂(Double-strandbreaks，DSBs)是DNA损伤最严重，修复过程最为复杂，对基因组稳定性威胁最大的损伤形式之一。如果DSBs没有及时修复，会影响遗传信息准确地传递，会诱发包括癌症在内的多种疾病，但另一面在肿瘤患者的放化疗过程中又会通过电离辐射或者化疗药物诱导癌细胞发生DSBs，促使癌细胞死亡。DNA双链断裂损伤发生后，细胞内一组高保守的PI3(Phosphoinositide 3)样激酶，包括ATM、ATR和DNA-PKcs被活化，然后磷酸化下游的几百个底物来调节细胞周期检查点和DNA损伤修复。
DNA-PKcs是非同源末端链接(Non-homologous end-joining，NHEJ)修复途径上游的关键蛋白，在DSBs发生时它能够迅速被环状异二聚体Ku70/Ku80募集到DNA断裂末端，形成DNA-PK复合物，激活DNA-PKcs的激酶活性。活化的DNA-PKcs可以磷酸化多种蛋白，如组蛋H2AX、Chk2、P53、53BP1、XRCC4、XLF、Ku70/80、Artemis等，它和ATM有很多共同底物，如H2AX和53BP1等，所以ATM与DNA-PKcs在DNA修复和胚胎发育发面有些重复的功能。
当DNA双链发生断裂时，DNA-PKcs标志性的翻译后修饰是其磷酸化，其磷酸化位点多达40多处并且呈集簇分布。研究最多的是Thr2609位点磷酸化。Thr2609主要被ATM和ATR磷酸化，Thr2609簇的磷酸化对于NHEJ很重要，因为通过丙氨酸取代消除这些位点的磷酸化会导致严重的放射敏感性，并降低体外的DNA末端连接能力。另外，Thr2609簇磷酸化和/或DNA-PKcs的构象改变有助于其与其他DNA修复分子的结合。
另一个特征明确的DNA-PKcs磷酸化簇是丝氨酸2056(Ser2056)簇。丝氨酸2056是体内对DSB的真正自磷酸化位点，Ser2056簇的磷酸化对于NHEJ很重要，因为该簇的缺失磷酸化会导致放射敏感性增加和DSB修复效率降低。
除了蛋白磷酸化，蛋白的泛素化是另外一个重要的发生在DNA损伤后的翻译后修饰形式。DNA双链断裂后，一组E2泛素交联酶和E3泛素连接酶包括UBC13，RNF8和RNF168被募集到DNA损伤位点并且泛素化染色体蛋白，比如组蛋白H2A和H2B。DNA双链断裂后，H2A在K13和K15赖氨酸位点被RNF168泛素化。RBX1蛋白的表达增加促使cullin1的neddylation修饰和其活性增加，cullin1是Skp1-Cullin1-Fbox泛素E3连接酶的一个关键组成部分。因此介导了EXO1在G1期的泛素化降解。DNA-PKcs活性的增加导致RBX1蛋白表达增加，并限制了DSB端ssDNA的形成，并抑制了G1细胞的HR修复。
肿瘤中DNA-PKcs相关的变化主要有prkdc基因突变及DNA-PKcs表达和活性的改变，而且不同肿瘤组织中其活性的变化也不尽相同。研究发现，结直肠癌和胶质瘤等活性增强，乳腺癌和宫颈癌中活性降低，食道癌和淋巴瘤中活性增强和降低的报道都有。增高的DNA-PKcs活性对于肿瘤来说是有利的。首先增高的活性使癌前细胞对DNA损伤的NHEJ修复能力提高，细胞逃脱死亡的命运。但这种修复易出错，引起DNA结构变化，促进肿瘤的形成。其次，肿瘤治疗中常用的DNA损伤性化疗药物和放疗发挥作用的主要机制就是造成DNA分子的致死性的双链断裂，进而诱导肿瘤细胞的死亡。而DNA-PKcs活性的增高能在一定程度上抑制细胞死亡，对放化疗产生耐受。此外，放化疗治疗后肿瘤组织中存活的细胞往往是DNA-PKcs活性高的细胞，它们对治疗不敏感，这也是疗效不好和预后差的原因。因此DNA-PKcs是肿瘤治疗的一个潜在靶点，对DNA-PKcs的检测和采用它的抑制剂来增强放化疗的效果是一种新的策略。
类泛素化(neddylation)修饰是由ATP依赖的ubiquitin类似的小分子蛋白Nedd8(neural precursor cell expressed,developmentally downregulated 8)经过激活酶E1、结合酶E2和连接酶E3的一系列催化作用后，使其C端甘氨酸与底物蛋白的赖氨酸残基相互作用发生共价结合的过程。DNA-PKcs可以在多个细胞周期相关的生化节点上调节DSBs的修复途径选择，但仅检测某些位点的磷酸化用于反应DNA-PKcs的活化状态及其生物学功能是不够的。由于目前对DNA-PKcs其他位点检测手段以及相关研究并未完善，对于DNA-PKcsK4007位点Neddylation修饰也存在大量空白。因此，制备一种DNA-PKcs蛋白K4007位点Neddylation修饰多克隆抗体对于评价DNA-PKcs对DNA断裂的修复功能极其在肿瘤中的作用具有重要意义。
发明内容
本发明根据DNA-PKcs蛋白序列，设计并合成类泛素化抗原多肽，之后用于动物免疫，制备多克隆抗体。
第一方面，本发明提供一种肿瘤DNA-PKcs蛋白的多克隆抗体，该多克隆抗体的抗原氨基酸序列如SEQ ID NO.1所示，
SEQ ID NO.1：CMDVFVLRGGKEPSFDWK。
第二方面，本发明提供一种肿瘤DNA-PKcs蛋白的多克隆抗体的制备方法，所述方法包括如下步骤：
S1.抗原多肽设计；
S2.用步骤S1设计的抗原免疫动物；
S3.在步骤S2免疫动物后，获取动物血清并进行纯化，获得本发明的肿瘤DNA-PKcs蛋白的多克隆抗体。
进一步的，所述抗原多肽设计是对DNA-PKcs蛋白进行类泛素化修饰，所述类泛素化修饰是指通过将类泛素化蛋白的C端羧基连接到DNA-PKcs蛋白的赖氨酸残基的ε-氨基上。
进一步的，所述进行类泛素化修饰蛋白选自LRGG、LALRGG、SUMO1、SUM02、SUM03、SUMO4、NEDD8、FAT10、FAT1、FUB1、UBL5、ISG15、UFM1、Urml、ATG12、ATG8、Apg12、MAP1LC3和GABARAPL中的一种或多种。
进一步，所述类泛素化蛋白优选为NEDD8。
第三方面，本发明提供一种如第一方面所述的肿瘤DNA-PKcs蛋白的多克隆抗体的药物组合物。
进一步的，所述药物组合物还可以包含佐剂和/或药学上可接受的辅料。
进一步的，所述组合物可以制备为疫苗、检测试剂或其他生物诊断试剂。
第四方面，本发明提供一种如第一方面所述的肿瘤DNA-PKcs蛋白的多克隆抗体在制备抑制肿瘤药物中的应用。
进一步的，所述肿瘤包括但不限于小细胞肺癌、非小细胞肺癌、卵巢癌、子宫内膜癌、乳腺癌、头颈癌、胸腺瘤、结直肠癌、胰腺癌、前列腺癌、膀胱癌或黑色素瘤。
附图说明
图1抗体开发路线及时间示意图；
图2Peptide 1质谱检测结果；
图3Peptide 2质谱检测结果；
图4Peptide 3质谱检测结果；
图5Peptide 4质谱检测结果；
图6抗体纯化示意图；
图7Dotblot检测结果图；
图8免疫荧光染色检测结果(红色点为DNA-PKcs K4007修饰信号，蓝色为DAPI染色)；
图9免疫荧光染色检测结果。
具体实施方式
本发明所用动物为健康新西兰兔3只。下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买得到。
实施例1抗原设计合成
定制抗体开发路线及时间如图1所示。
1、抗原的设计
DNA-PKcs蛋白通过类泛素化蛋白NEDD8进行类泛素化修饰，即将NEDD8分子C端羧基连接到DNA-PKcs蛋白序列第4007位赖氨酸位点的ε-氨基上，在4007位置形成了类泛素化序列LRGG。
根据类泛素化修饰的DNA-PKcs蛋白的序列信息，在4007位点左右分别选取不超过10个氨基酸的多肽，设计并合成2条修饰多肽，2条非修饰多肽，多肽信息如下表1所示。
表1多肽信息表
2、质谱检测
对合成的4条多肽的序列进行抗原质谱检测。将合成的肽段混合物，在质谱仪中电离形成带电离子，质谱分析器的电场、磁场将具有特定质量与电荷比值(即质荷比，M/Z)的肽段离子分离开来，经过检测器收集分离的离子，确定每个离子的M/Z值。经过质量分析器可分析出每个肽段的M/Z，得到蛋白质所有肽段的M/Z图谱，即蛋白质的一级质谱峰图。离子选择装置自动选取强度较大肽段离子进行二级质谱分析，输出选取肽段的二级质谱峰图，通过和理论上蛋白质经过胰蛋白酶消化后产生的一级质谱峰图和二级质谱峰图进行比对而鉴定本发明所需要的抗原。
将合成多肽通过质谱检测，且质谱检测结果合格。4条多肽的序列准确，4条肽段的实测质量和理论质量的差别在10ppm之内，氨基酸序列无误(见图2-5所示，坐标轴纵轴表示离子峰的强度，横轴表示质量和电荷的比率)。
实施例2实验动物的免疫
将2条修饰多肽与KLH偶联，用于兔子免疫。
用生理盐水稀释免疫原，然后与相应的弗氏佐剂进行1：1混合。抗原和佐剂完全混合，形成稳定的乳剂，用注射器抽取抗原混合物，于兔子双肩皮下两点和双后腿肌肉两点注射抗原，每个区域大约1/4体积的免疫原。这样免疫原可以持久存在从而提高免疫应答。每只兔子共免疫4次，分别在第1天，第21天，第28天，第35天。
取血第1次：在第45天，取全血30mL并进行离心，离心后取上清，送实验室进行血清筛选检测，检测内容包括ELISA；取血第2/3/4次：在第50天，第65天，第70天分3次取全血，每次20mL，离心后取上清，送实验室进行血清筛选检测。
实施例3ELISA法测定抗体的校价
用抗原多肽peptide1和peptide2各免疫2只SPF实验级新西兰大白兔，peptide1免疫的兔子标识为R1、peptide2免疫的兔子标识为R2，经过多次免疫后，分别进行ELISA初筛，初步评估抗血清的滴度和特异性。
3.1血清初筛ELISA检测
在包被了抗原修饰性多肽、或者非修饰性对照多肽的96孔酶标板中，依不同的稀释比例加入抗血清孵育，之后施加酶标二抗以及TMB显色底物，检测多肽和抗血清的结合。在450nm波长的吸光度(OD450)为1左右时，所对应的抗血清稀释比为抗血清的滴度。血清滴度用灰色阴影标出。
ELISA检测结果如下表2所示，R1兔血清识别修饰性多肽1的滴度在1:54K左右，识别修饰性多肽2的滴度在1:6K左右，识别非修饰性多肽3的滴度在1:6K左右，基本不识别非修饰多肽4。R2兔血清识别修饰性多肽1和修饰性多肽2的滴度在1:54K-1:486K之间，识别非修饰性多肽3的滴度在1:486K之间，基本不识别非修饰多肽4。总的来说，R1和R2兔子的抗血清均呈现很好的滴度，都进行纯化。
表2血清ELISA检测结果
实施例4:亲和层析柱纯化抗体
4.1准备proteinA亲和柱：
通常选取5mL或10mLproteinA填料，将等体积的填料和PBS缓冲溶液混合、搅拌，抽气去除填料中的气泡。将proteinA填料缓慢加入玻璃柱中，灌制层析柱。此过程中要避免柱干。灌注完毕后用10倍体积预冷的PBS缓冲溶液平衡柱子。
4.2ProteinA亲和层析：
4.2.1将血清用过滤器进行过滤后，上样到平衡好的proteinA层析柱上，为检测抗血清与填料的结合效率，需保留上样流出液。
4.2.2用PBS缓冲溶液清洗柱子，再用150mM甘氨酸缓冲液进行洗脱。收集洗脱液，并加入中和缓冲溶液调制pH为7。
4.2.3将proteinA纯化后得到的粗纯IgG上样到平衡好的抗原多肽亲和层析柱上，专一性的富集目的抗体。
4.2.4去除非特异性抗体：
将上一步得到的目的抗体上样到平衡好的吡啶甲酰化修饰和异烟酸酰化修饰亲和层析柱上，直接收集流出液，从而去除非特异性的抗体成分。
4.2.5抗体保存：
测定蛋白质的含量。加入10％的甘油以便保存抗体，将纯化的抗体分装后在2℃-8℃保存。
抗体纯化示意图如图6所示，将血清用过滤器进行过滤后，上样到平衡好的protein A层析柱上，得到ET1，将ET1上样到Antigen Peptides层析柱上，得到ET2，将ET2上样到non modified control peptides层析柱上，得到FT3。
实施例5:ELISA进行抗体质控检测
取足够量的兔血清，进行多步亲和纯化。纯化后的抗体分别进行ELISA和Dot Blot检测。
5.1ELISA检测：
5.1.1抗原包被：用包被液将抗原稀释，按照50ug/孔的量依次加入酶标板，冰箱4℃过夜或37℃烘箱孵育2h。
5.1.2洗板：将前一天包被好的酶标板取出，加入1×TBST洗涤3次。
5.1.3封闭：将清洗后的酶标板加入1％BSA封闭液，37℃孵育1h后洗涤1-3次。
5.1.4一抗孵育：将抗体按照1:1K开始做3倍梯度稀释。根据实际情况调节稀释体积。依次加入酶标板中，37℃温育1.5h后洗涤1-3次。
5.1.5二抗孵育：用1％BSA封闭液将二抗稀释到1：10K,室温或37℃孵育45min后洗涤1-3次。
5.1.6显色：加入TMB显色液5-10min后1M硫酸终止显示反应，用酶标仪读取数据。
从R1和R2兔子血清纯化出的抗体标识为Ab1和Ab2(Ab2强识别于非修饰性多肽3舍去)。ELISA检测结果如表3所示：Ab1抗体识别修饰多肽1的滴度约1:1458K，不识别非修饰性多肽3和4。
表3抗体ELISA检测结果
实施例6:Dot Blot进行抗体质控检测
6.2Dotblot检测：
6.2.1点样：按照1ng,4ng,16ng,64ng梯度将未交联抗原多肽点到PVDF膜上。
6.2.2封闭：待膜表面干燥后，加入封闭液，室温封闭60min。
6.2.3洗涤：用1×TBST洗涤10min。
6.2.4一抗孵育：用2.5-5％的脱脂奶粉将抗体稀释，室温孵育2h后用1×TBST洗涤3次,每次5-10min。
6.2.5二抗孵育：根据一抗抗体属性选择对应的鼠抗或兔抗；将二抗按照1：10K稀释比加入，室温孵育45min-1h后，用1×TBST洗涤3次,每次5-10min。
6.2.6将洗涤后的膜加显色底物后曝光。
结果如图7所示，随着上样量由1ng逐渐递增到64ng，Dot blot实验结果的灰度值逐渐递增，表明所纯化抗体构建成功并且可用于实验室监测。
实施例7:免疫荧光染色进行抗体质控检测
7.1BEAS-2B细胞消化计数后在玻璃爬片上培养贴壁24小时
7.2用10μM VP-16处理BEAS-2B细胞4小时，VP-16对细胞内DNA造成损伤。
7.3细胞除去就培养基，PBS洗两遍后，4％多聚甲醛室温固定15分钟
7.4PBS洗一遍后用0.5％Triton-X100打孔10分钟。
7.510％FBS常温封闭1小时。
7.6用DNA-PKcs K4007位点抗体1：500稀释，与细胞爬片室温孵育1小时。
7.7PBS洗三遍去除非特异结合。
7.8用罗丹明TRITC标记的羊抗兔二抗室温孵育30分钟。
7.9PBS洗三遍去除非特异结合。
7.10用ProLong Gold antifade封片剂封片(内含细胞核染料DAPI)。
7.11共聚焦显微镜进行观察并统计分析结果。
结果如图8所示，10μM VP-16处理BEAS-2B细胞4小时后，DNAPKcs K4007位点neddylation修饰抗体能够识别DNA-PKcs neddylation修饰形成的foci。
实施例8:免疫沉淀-Western blot进行抗体质控检测
8.1培养BEAS-2B细胞，用10μM VP-16处理BEAS-2B细胞。
8.2分别在0h、1h、4h、8h收集VP-16处理的BEAS-2B细胞。
8.3BEAS-2B细胞用RIPA裂解液裂解。
8.4裂解蛋白进行BCA定量。
8.5用DNA-PKcs抗体分别与不同时间点的蛋白进行4度孵育过夜。
8.6加入ProteinA/G柱料继续孵育4h。
8.7离心，弃上清。裂解液洗三遍。
8.8加入5X上样缓冲液，95度加热5分钟进行蛋白变性。
8.9每组蛋白进行SDS-PAGE电泳分离。
8.10将PAGE胶分离后的蛋白进行转膜结合到PVDF膜。
8.115％脱脂牛奶对PVDF进行封闭1小时。
8.12分别将PVDF膜与DNA-PKcs K4007修饰抗体和DNA-PKcs抗体孵育。
8.13TBST洗三遍后，进行HRP标记的二抗孵育。
8.14加入HRP底物，曝光显影。
结果如图9所示，10μM VP-16处理BEAS-2B细胞后，随着DNA损伤时间的延长，DNA-PKcs K4007位点neddylation修饰随时间增强。
Claims (10)
1.一种肿瘤DNA-PKcs蛋白的多克隆抗体，该多克隆抗体的抗原氨基酸序列如SEQ IDNO.1所示，
SEQ ID NO.1：CMDVFVLRGGKEPSFDWK。
2.一种肿瘤DNA-PKcs蛋白的多克隆抗体的制备方法，所述方法包括如下步骤：
S1.抗原多肽设计；
S2.用步骤S1设计的抗原免疫动物；
S3.在步骤S2免疫动物后，获取动物血清并进行纯化，获得本发明的肿瘤DNA-PKcs蛋白的多克隆抗体。
3.如权利要求2所述的肿瘤DNA-PKcs蛋白的多克隆抗体的制备方法，其特征在于，所述抗原多肽设计是对DNA-PKcs蛋白进行类泛素化修饰，所述类泛素化修饰是指通过将类泛素化蛋白的C端羧基连接到DNA-PKcs蛋白的赖氨酸残基的ε-氨基上。
4.如权利要求2所述的肿瘤DNA-PKcs蛋白的多克隆抗体的制备方法，其特征在于，所述进行类泛素化修饰蛋白选自LRGG、LALRGG、SUMO1、SUM02、SUM03、SUMO4、NEDD8、FAT10、FAT1、FUB1、UBL5、ISG15、UFM1、Urml、ATG12、ATG8、Apg12、MAP1LC3和GABARAPL中的一种或多种。
5.如权利要求4所述的肿瘤DNA-PKcs蛋白的多克隆抗体的制备方法，其特征在于，所述类泛素化蛋白选自NEDD8。
6.一种如权利要求1所述的肿瘤DNA-PKcs蛋白的多克隆抗体的药物组合物。
7.如权利要求6所述的肿瘤DNA-PKcs蛋白的多克隆抗体的药物组合物，其特征在于，所述药物组合物还可以包含佐剂和/或药学上可接受的辅料。
8.如权利要求6所述的肿瘤DNA-PKcs蛋白的多克隆抗体的药物组合物，其特征在于，所述组合物可以制备为疫苗、检测试剂或其他生物诊断试剂。
9.一种如权利要求1所述的肿瘤DNA-PKcs蛋白的多克隆抗体在制备抑制肿瘤药物中的应用。
10.如权利要求9所述的肿瘤DNA-PKcs蛋白的多克隆抗体在制备抑制肿瘤药物中的应用，其特征在于，所述肿瘤选自小细胞肺癌、非小细胞肺癌、卵巢癌、子宫内膜癌、乳腺癌、头颈癌、胸腺瘤、结直肠癌、胰腺癌、前列腺癌、膀胱癌或黑色素瘤或黑色素瘤。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311664592.2A CN117586406A (zh) | 2023-12-06 | 2023-12-06 | 类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311664592.2A CN117586406A (zh) | 2023-12-06 | 2023-12-06 | 类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117586406A true CN117586406A (zh) | 2024-02-23 |
Family
ID=89914999
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311664592.2A Pending CN117586406A (zh) | 2023-12-06 | 2023-12-06 | 类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117586406A (zh) |
-
2023
- 2023-12-06 CN CN202311664592.2A patent/CN117586406A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2321197T3 (es) | Fosforilacion de proteinas en rutas de señalizacion de c-src. | |
| JP2003528296A (ja) | 質量に基づく分離を含む疾患マーカーの同定 | |
| AU2008203968A1 (en) | Peptide markers for diagnosis of angiogenesis | |
| CN107163104B (zh) | 核酸适配体-多肽复合物探针及其制备方法和应用 | |
| JP6060425B2 (ja) | 膀胱癌の診断 | |
| KR20130103547A (ko) | 모에신 단편 및 그의 용도 | |
| US20090298093A1 (en) | Reagents for the Detection of Protein Phosphorylation in ATM & ATR Kinase Signaling Pathways | |
| JP2016222725A (ja) | モノクローナル抗体を生成し、検証し、そして使用するための方法およびシステム | |
| Liu et al. | A photocleavable peptide-tagged mass probe for chemical mapping of epidermal growth factor receptor 2 (HER2) in human cancer cells | |
| EP1907842B1 (en) | Identification of non-small cell lung carcinoma (nsclc) tumors expressing pdgfr-alpha | |
| Wei et al. | Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation | |
| WO2009072728A1 (en) | Method for diagnosis of disease using quantitative monitoring of protein tyrosine phosphatase | |
| KR101571940B1 (ko) | 면역성 혈소판 감소증과 관련된 모에신 단편 | |
| US20170138958A1 (en) | Method for measuring anti-wt1 antibody | |
| AU2004296643B2 (en) | Use of enzymatic inhibitors of h-PRUNE for the prevention and treatment of the metastases of tumours overexpressing h-PRUNE | |
| CN117586406A (zh) | 类泛素化修饰的DNA-PKcs蛋白的多克隆抗体及其制备方法 | |
| CN104364374A (zh) | 癌的检测方法及识别胰脏特异性的核糖核酸酶1的抗体 | |
| US20100151495A9 (en) | Reagents for the detection of protein phosphorylation in carcinoma signaling pathways | |
| KR102320436B1 (ko) | 교모종줄기세포 엑소좀 유래 펩타이드를 포함하는 교모세포종의 진단용 바이오마커 조성물 | |
| US7718780B2 (en) | Preparation of monoclonal antibody to N-myc downstream regulated gene 2 and determination of NDRG2 using protein chip | |
| CN110950958B (zh) | 一种抗brca2单克隆抗体及其应用 | |
| KR101240199B1 (ko) | 대장암 줄기세포 선별용 단백질성 마커 | |
| JPH07503141A (ja) | 新規抗▲ｒｂ▼モノクローナル抗体の特性 | |
| KR101231235B1 (ko) | 전립선암 줄기세포 선별용 단백질성 마커 | |
| CN101891813A (zh) | 白血病耐药细胞膜蛋白及其作为耐药靶蛋白的用途 |
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 |