CN117659187A - Anti-human NUT protein rabbit monoclonal antibody and application thereof - Google Patents
Anti-human NUT protein rabbit monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN117659187A CN117659187A CN202311712149.8A CN202311712149A CN117659187A CN 117659187 A CN117659187 A CN 117659187A CN 202311712149 A CN202311712149 A CN 202311712149A CN 117659187 A CN117659187 A CN 117659187A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- acid sequence
- monoclonal antibody
- seq
- rabbit monoclonal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 50
- 108010003571 Nut Proteins Proteins 0.000 title abstract description 32
- 238000013115 immunohistochemical detection Methods 0.000 claims abstract description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 35
- 210000001519 tissue Anatomy 0.000 claims description 34
- 101001122114 Homo sapiens NUT family member 1 Proteins 0.000 claims description 29
- 150000001413 amino acids Chemical class 0.000 claims description 23
- 206010028980 Neoplasm Diseases 0.000 claims description 19
- 102000057037 human NUTM1 Human genes 0.000 claims description 13
- 210000004881 tumor cell Anatomy 0.000 claims description 10
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 8
- 210000002741 palatine tonsil Anatomy 0.000 claims description 7
- 210000001685 thyroid gland Anatomy 0.000 claims description 7
- 206010033701 Papillary thyroid cancer Diseases 0.000 claims description 6
- 210000001550 testis Anatomy 0.000 claims description 6
- 208000030045 thyroid gland papillary carcinoma Diseases 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 108091033319 polynucleotide Proteins 0.000 claims description 5
- 239000002157 polynucleotide Substances 0.000 claims description 5
- 102000040430 polynucleotide Human genes 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 102000003992 Peroxidases Human genes 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 4
- 230000037430 deletion Effects 0.000 claims description 4
- 210000000496 pancreas Anatomy 0.000 claims description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 238000011191 terminal modification Methods 0.000 claims description 4
- 201000009030 Carcinoma Diseases 0.000 claims description 3
- 206010020751 Hypersensitivity Diseases 0.000 claims description 3
- 239000002981 blocking agent Substances 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims 1
- 239000012192 staining solution Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 102100027086 NUT family member 1 Human genes 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- 201000011510 cancer Diseases 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 238000000034 method Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000004927 fusion Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 210000004923 pancreatic tissue Anatomy 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 230000002381 testicular Effects 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101100406146 Homo sapiens NUTM1 gene Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 101150111780 NUTM1 gene Proteins 0.000 description 2
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 101100326315 Homo sapiens BRD4 gene Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 101710137446 NUT family member 1 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000018732 detection of tumor cell Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000000920 spermatogeneic effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application belongs to the technical field of immunodetection, and discloses an anti-human NUT protein rabbit monoclonal antibody and application thereof. The application also provides an immunohistochemical detection kit containing the anti-human NUT protein rabbit monoclonal antibody and application of the antibody or the kit in the immunohistochemical detection, and the anti-human NUT protein rabbit monoclonal antibody has higher specificity and sensitivity, can be specifically combined with NUT protein, and remarkably improves the specificity and reliability of the immunodetection NUT protein.
Description
Technical Field
The application relates to the technical field of immunodetection, in particular to an anti-human NUT rabbit monoclonal antibody and application thereof.
Background
Testis nucleoprotein (Nuclear protein in testis, NUT), expressed by the NUTM1 gene (at 15q 14), is expressed in sperm cells after testicular meiosis under physiological conditions. When the NUTM1 gene fuses with genes involved in transcriptional regulation, it can become an oncogenic driver to form tumors, namely, NUT cancers, which usually occur in the midline of the chest, head and neck, also known as midline cancers. A variety of non-midline organs are now being diagnosed, such as lung (1/2), head and neck (1/3), thymus, bladder, liver/pancreas, brain, and other tissues have also been reported. Midline cancer is mostly in its original form, and in most cases has no specific structure, consists of homogeneous tumor cells, is single in morphology, is undifferentiated, and has a higher nucleus and active mitosis.
Whole genome sequencing found that NUT cancer may be driven by a single NUTM1 fusion gene, few other oncogene mutations, cross-translocations of the NUT gene with other genes to form a NUT-variant fusion gene, expressed fusion proteins capable of significantly inhibiting squamous cell differentiation and driving cancer genesis, about 70-80% of cases found translocation of the NUT gene with the 19p13.1 BRD4 gene to form a BRD4-NUTM1 fusion gene, BRD3-NUTM1 fusion (about 15-20%) and NSD3-NUTM1 fusion (about 6%), and somatic genetic alterations were the basis for the pathogenesis of midline cancer. At present, FISH with a NUT 1 breakpoint 15q14 probe is considered as a gold standard for diagnosis, however, 100% hybridization cannot be achieved, some NUT breakpoints cannot be detected through the probe, judging specificity is strong, only NUT gene rearrangement can be detected, specific chaperones cannot be determined, and detection cost is high.
Almost 100% of midline cancer cases express NUT nucleoprotein (more than or equal to 50% of tumor cells are all expressed), and the specificity and the sensitivity are over 90%, so that monoclonal antibodies with strong specificity and high sensitivity are developed to be used for diagnosing midline cancer by an immunohistochemical method, the detection cost is reduced, and the detection accuracy can be improved by combining a FISH method.
Disclosure of Invention
In order to overcome the technical problems, the application provides the anti-human NUT rabbit monoclonal antibody with high sensitivity, strong specificity and high affinity and the application thereof in an immunohistochemical detection kit, thereby providing assistance for diagnosis of the midline cancer related to the human NUT.
In a first aspect, the present application provides a rabbit monoclonal antibody against human NUT protein, the rabbit monoclonal antibody comprising a light chain variable region (VL) comprising LCDR1-3, the LCDR1 comprising an amino acid sequence as shown in SEQ ID No.4, the LCDR2 comprising an amino acid sequence as shown in SEQ ID No.5, and the LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 6.
Further, the rabbit monoclonal antibody further comprises a heavy chain variable region (VH) comprising HCDR1-3, the HCDR1 comprising an amino acid sequence of SEQ ID No.8, the HCDR2 comprising an amino acid sequence of SEQ ID No.9, and the HCDR3 comprising an amino acid sequence of SEQ ID No. 10.
Further, the light chain variable region (VL) comprises an amino acid sequence shown as SEQ ID No.3, or comprises an amino acid sequence having more than 90% homology, preferably, an amino acid sequence having 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology, obtained by substitution, deletion and/or addition of one or more amino acids and/or terminal modification of any one or more amino acids in the amino acid sequence shown as SEQ ID No. 3.
Further, the light chain variable region (VL) has a total length of 106 amino acids, the FR has 4 domain amino acids of 26, 17, 36 and 10, the CDR has 3 domain amino acids of 6, 3 and 8, and the LCDR1, LCDR2 and LCDR3 have regions of 27-32aa, 50-52aa, 89-96aa, respectively, and the amino acid sequences thereof are: QSISSR (SEQ ID NO. 4); QAS (SEQ ID NO. 5); QSYYYYTAS (SEQ ID NO. 6).
Further, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence having more than 90% homology, preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology, obtained by substitution, deletion and/or addition of one or more amino acids and/or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO. 7.
Further, the heavy chain variable region (VH) comprises 110 amino acids, the FR of which has a number of 4 domain amino acids of 24, 17, 36 and 11, respectively; the amino acid numbers of 3 domains of the CDR are 8, 7 and 7 respectively, the corresponding amino acid regions of HCDR1, HCDR2 and HCDR3 are 25-32aa, 50-56aa and 93-99aa respectively, and the sequence is GFTINSYH (SEQ ID NO. 8); ISDGATY (SEQ ID NO. 9); ASTMYN (SEQ ID NO. 10).
By adopting the technical scheme, the monoclonal antibody of the anti-human NUT protein can be specifically combined with the NUT protein, and the specificity, accuracy and reliability of the NUT protein immunodetection are obviously improved.
In a second aspect, the present application also provides an isolated polynucleotide encoding the rabbit monoclonal antibody of the first aspect.
In a third aspect, the present application also provides a recombinant expression vector comprising a polynucleotide according to the second aspect of the present application.
By adopting the technical scheme, the rabbit monoclonal antibody of the anti-human NUT protein can be efficiently prepared in vitro through the separated polynucleotide and the recombinant expression vector, and the technical effects of short preparation period and high utilization efficiency can be achieved by utilizing a molecular recombination method to prepare the antibody, and meanwhile, the editing function of the downstream of the antibody can be realized.
In a fourth aspect, the application also provides an application of the rabbit monoclonal antibody in the first aspect in preparing an immunohistochemical detection kit for marking human NUT protein in normal tissues and pathological tissues.
Further, the normal tissue includes testis, tonsil, thyroid, pancreas, and the lesion tissue includes tumor tissue including midline cancer tumor cells and papillary thyroid cancer tumor cells.
By adopting the technical scheme, the rabbit monoclonal antibody of the anti-human NUT protein can be used as a primary antibody for marking the NUT protein in normal combination and tumor tissues, and the specificity and reliability of the NUT protein immunodetection are obviously improved.
In a fifth aspect, the present application also provides an immunohistochemical detection kit for human NUT protein, the immunohistochemical detection kit comprising a primary antibody, the primary antibody comprising the monoclonal antibody of the first aspect.
Further, the kit also comprises an antigen retrieval liquid, an endogenous peroxidase blocking agent, a hypersensitive secondary antibody reagent, a DAB substrate buffer solution, a DAB concentrated color development liquid and a hematoxylin staining liquid.
By adopting the technical scheme, IHC detection of NUT protein in normal tissues, tumor and other pathological tissues can be realized more conveniently.
Compared with the prior art, the monoclonal antibody of the anti-human NUT protein can be specifically combined with the NUT protein, and the specificity, accuracy and reliability of the NUT protein immunodetection are obviously improved. Meanwhile, the immunohistochemical detection kit provided by the application adopts the monoclonal antibody of the anti-human NUT protein as a primary antibody, can realize the marking of the human NUT protein in pathological tissues such as normal tissues, tumors and the like, can meet the requirements of pathologists on the anti-NUT monoclonal antibody, and can provide guarantee for the accurate diagnosis of pathologists.
Drawings
FIG. 1 is a graph showing the results of immunohistochemical detection of NUT protein expression in testis, tonsil, thyroid gland, pancreas as described in examples 4-7, wherein primary antibody used for hybridization is rabbit monoclonal antibody OTIR6H1 against human NUT protein, wherein figures a-b are testis tissue and figures c-e are tonsil, thyroid gland, pancreas tissue, respectively.
FIG. 2 is a graph showing the results of immunohistochemical detection of NUT protein expression in midline carcinoma and papillary thyroid carcinoma as described in examples 8-9, wherein primary antibody used for hybridization is rabbit monoclonal antibody OTIR6H1 against human NUT protein, wherein figure a is midline carcinoma tissue and figure b is papillary thyroid carcinoma tissue.
Detailed Description
The present application is further illustrated below in conjunction with specific embodiments, it being understood that these embodiments are merely illustrative of the present application and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions, those described in the laboratory manual, or those suggested by the manufacturer.
EXAMPLE 1 preparation of anti-NUT Rabbit monoclonal antibodies
1. Immunization of animals
Amino acids 957-976 and 1109-1128 of the synthesized NUT protein are used as detection materials for magnetic bead screening and ELISA screening, the amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences are respectively coupled with KLH and used as immunogens. The Freund's adjuvant is used for emulsification, about 2kg of New Zealand white rabbits are immunized by subcutaneous injection, the immunization dose is 500 mug/rabbit, the second immunization is carried out after two weeks, the incomplete Freund's adjuvant is used for emulsification, and the immunization dose is 250 mug/rabbit. Tail blood is taken after three times of immunization, and serum titer is measured by ELISA and IHC method gradient dilution; based on the ELISA titers 128000 with an OD450 of greater than 1.0 and the intensity of the nuclear stain signal detected on the tissue by IHC, it was determined whether PBMCs were collected or continued immunization.
2. PBMCs isolation, specific B cell sorting and cloning recombination
Fixing the immunized New Zealand white rabbits with standard serum detection titer on an operating table, shaving the furs of heart parts, wiping and sterilizing skin by alcohol cotton balls, selecting the most obvious heart beat part, puncturing by a 50ml syringe, flushing blood into the syringe after a needle penetrates into the heart, rapidly pulling out the needle after the required blood volume is obtained, transferring whole blood in the syringe into a sterile 50ml tube, uniformly mixing with equal amount of PBS, slowly adding dropwise above lymphocyte separation liquid, centrifuging at room temperature of 400 Xg for 30min, and dividing the liquid level into four layers from top to bottom: yellow plasma layer, white thin film layer (i.e. mononuclear cell layer), separated liquid layer and erythrocyte layer. The rabbit PBMCs were obtained by carefully pipetting the mononuclear cell layer and washing with PBS to remove platelet and lymphocyte isolates. From this, antigen-specific B cells were continuously sorted and cultured, and the synthetic NUT peptides corresponding to amino acids 957 to 976 and amino acids 1109 to 1128 were mixed and coated, and positive clones were selected by ELISA. Positive clones are lysed, lysate is collected, RNA is extracted from the positive clones, the RNA is reversely transcribed into cDNA, the full-length sequence of the light and heavy chains of the naturally paired rabbit monoclonal antibodies is amplified from the cDNA of the corresponding positive clone, a rabbit monoclonal antibody expression vector is constructed by a cloning recombination method, and the sequence is determined by sequencing.
3. Preparation and purification of monoclonal antibodies
In order to obtain a rabbit monoclonal antibody capable of specifically recognizing human NUT protein, the invention loads heavy chain and light chain genes of the rabbit monoclonal antibody onto an expression vector, and transfects a recombinant plasmid into HEK293 cells; the supernatant was incubated 120-144h after transfection with recombinant rabbit monoclonal antibodies recognizing human NUT protein. Collecting cell suspension, centrifuging to obtain supernatant, performing antibody purification by affinity chromatography, determining rabbit monoclonal antibody OTIR6H1 as human NUT protein, and measuring the concentration of the purified monoclonal antibody by BCA method.
EXAMPLE 2 analysis of the variable region Gene and amino acid sequence of Rabbit monoclonal antibody OTIR6H1
And respectively taking recombinant plasmids of the OTIR6H1 antibody as DNA templates, designing a light chain variable region and a heavy chain variable region sequencing primer according to the 5' -end carrier sequences of the light chain and the heavy chain on the templates, and sequencing by adopting a sequencer ABI 3730. The nucleotide sequence of the rabbit monoclonal antibody OTIR6H1 light chain variable region is obtained by sequencing. And (3) carrying out sequencing result data analysis on the nucleotide sequences of the light chain and the heavy chain by using IMGT/V-QUEST analysis software on http:// www.imgt.org to obtain the variable region amino acid sequence of the light chain of the rabbit monoclonal antibody OTIR6H1, wherein the variable region amino acid sequence of the light chain is shown as SEQ ID NO.3, and the variable region amino acid sequence of the heavy chain is shown as SEQ ID NO. 4.
The total length of the light chain variable region (VL) is 106 amino acids, the number of 4 domain amino acids of the FR is 26, 17, 36 and 10 respectively, the number of 3 domain amino acids of the CDR is 6, 3 and 8 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27-32aa, 50-52aa, 89-96aa respectively, the amino acid sequences of which are: QSISSR (SEQ ID NO. 5); QAS (SEQ ID NO. 6); QSYYYYTAS (SEQ ID NO. 7).
The heavy chain variable region (VH) contains 110 amino acids, the FR of which has 4 domain amino acid numbers of 24, 17, 36 and 11, respectively; the amino acid numbers of 3 domains of the CDR are 8, 7 and 7 respectively, the corresponding amino acid regions of HCDR1, HCDR2 and HCDR3 are 25-32aa, 50-56aa and 93-99aa respectively, and the sequence is GFTINSYH (SEQ ID NO. 8); ISDGATY (SEQ ID NO. 9); ASTMYN (SEQ ID NO. 10).
Example 3 immunohistochemical detection kit containing anti-human NUT protein Rabbit monoclonal antibody OTIR6H1
An immunohistochemical detection kit containing an anti-human NUT protein rabbit monoclonal antibody OTIR6H1 comprises antigen retrieval liquid [1mM EDTA,10mM Tris buffer (pH8.0) ], the anti-human NUT protein rabbit monoclonal antibody OTIR6H1 (0.17 mug/ml) obtained by preparation and purification of example 1, endogenous peroxidase blocking agent (3% hydrogen peroxide), a hypersensitive enzyme-labeled goat anti-mouse/rabbit IgG polymer, DAB color development liquid and hematoxylin staining liquid.
EXAMPLE 4 IHC detection of testis tissue by rabbit monoclonal antibody OTIR6H1
Testicular tissue was tested using the immunohistochemical test kit of example 3 as follows:
(1) Formalin-fixed testicular tissue blocks were paraffin embedded and sectioned using a Leica tissue microtome to a tissue thickness of 4 μm.
(2) Dewaxing and hydration: analytically pure xylene for 10min3 times, absolute ethanol for 10min3 times, 95% ethanol for 5min,85% ethanol for 5min,75% ethanol for 5min, deionized water for 3min3 times.
(3) Adding antigen retrieval liquid into the autoclave for autoclave retrieval for 2.5min, opening the autoclave when the temperature of the autoclave is reduced to about 90 ℃, taking out the specimen, naturally cooling to room temperature, and soaking in deionized water for 3min for 3 times.
(4) Inactivating tissue endogenous peroxidase by using 3% hydrogen peroxide, standing at room temperature for 10min, and soaking in deionized water for 5min for 3 times.
(5) anti-NUT rabbit monoclonal antibody OTIR6H1 (0.17. Mu.g/ml) was added and placed in a wet box, incubated at 37℃for 60min, and washed with PBST (0.1% Tween-20) for 5min3 times.
(6) A supersensitive enzyme-labeled goat anti-mouse/rabbit IgG polymer (Catalog No. PV-8000) was added dropwise, incubated at 37℃for 30min, and washed with PBS for 5min3 times.
(7) The DAB solution is used for developing color for 5min, and distilled water is used for washing.
(8) Hematoxylin counterstains the cell nuclei for 2min, distilled water is used for rinsing, 1% hydrochloric acid is differentiated, distilled water is used for rinsing 3 times, and standing is carried out at room temperature for 1min.
(9) Dehydration and transparency: 75% ethanol 5min,85% ethanol 5min,95% ethanol 5min,100% ethanol 5min×3 times; xylene 5min×3 times, neutral resin seals.
(10) And (5) microscopic examination.
The results are shown in figures 1 a-b, b is an enlarged view of the red frame position in figure a, the arrow indicates spermatogenic cells, the nuclei are positively stained, the triangle labeled support cells are unstained, and the cells are negatively stained.
EXAMPLE 5 IHC detection of tonsil tissue by rabbit monoclonal antibody OTIR6H1
The specific procedure for the detection of tonsil tissue using the immunohistochemical detection kit of example 3 was the same as in example 4, and the results are shown in figure 1, panel c, where all cell types of tonsil tissue were negative, i.e., were not expressing NUT protein.
EXAMPLE 6 IHC detection of thyroid tissue by rabbit monoclonal antibody OTIR6H1
The immunohistochemical detection kit in example 3 is used for detecting thyroid tissues, the specific steps are the same as in example 4, and the result is shown in a d diagram in fig. 1, and all cell types of thyroid tissues are negative, namely, NUT protein is not expressed.
EXAMPLE 7 IHC detection of pancreatic tissue by the rabbit monoclonal antibody OTIR6H1
Pancreatic tissue was examined using the immunohistochemical detection kit of example 3, and the specific procedure was the same as in example 4, and the results were shown in FIG. 1, e, in which all cell types of pancreatic tissue were negative, i.e., NUT protein was not expressed.
Example 8 IHC detection of tumor cell tissue of midline cancer by rabbit monoclonal antibody OTIR6H1
The immunohistochemical detection kit in example 3 is adopted to detect the tumor cell tissues of the central line cancer, the specific steps are the same as those in example 4, the result is shown as a graph in fig. 2a, the cell nuclei of the tumor cells are positive, the lymphocyte in the interstitium and the like are negative, and the expression profile of the NUT is consistent.
EXAMPLE 9 IHC detection of thyroid papillary carcinoma tumor cell tissue by rabbit monoclonal antibody OTIR6H1
The immunohistochemical detection kit in the embodiment 3 is adopted to detect the cell tissues of the papillary thyroid cancer, the specific steps are the same as those in the embodiment 4, and the result is shown as a graph b in fig. 2, and both tumor cells and interstitial cells show negative expression and accord with the expression profile of NUT.
In conclusion, IHC results show that the rabbit monoclonal antibody OTIR6H1 of the anti-human NUT protein has good specificity, has specific nuclear staining on tissues and tumors in which the NUT protein is expressed, has no nonspecific staining on tissues in which the NUT is not expressed, and proves that the rabbit monoclonal antibody OTIR6H1 of the anti-human NUT protein has good specificity, has clear staining intensity at the concentration of 0.17 mug/ml and has better sensitivity. The rabbit monoclonal antibody OTIR6H1 of the anti-human NUT protein can completely meet the requirements of pathologists on the anti-NUT monoclonal antibody, and can provide guarantee for the accurate diagnosis of pathologists.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. A rabbit monoclonal antibody against human NUT protein, comprising a light chain variable region (VL) comprising LCDR1-3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID No.4, wherein LCDR2 comprises an amino acid sequence as shown in SEQ ID No.5, and wherein LCDR3 comprises an amino acid sequence as shown in SEQ ID No. 6.
2. The rabbit monoclonal antibody of claim 1, further comprising a heavy chain variable region (VH) comprising HCDR1-3, the HCDR1 comprising an amino acid sequence as shown in SEQ ID No.8, the HCDR2 comprising an amino acid sequence as shown in SEQ ID No.9, and the HCDR3 comprising an amino acid sequence as shown in SEQ ID No. 10.
3. The rabbit monoclonal antibody according to claim 1, wherein the light chain variable region (VL) comprises an amino acid sequence as shown in SEQ ID No.3, or comprises an amino acid sequence having more than 90% homology obtained by substitution, deletion and/or addition of one or more amino acids and/or terminal modification of any one or more amino acids of the amino acid sequence shown in SEQ ID No. 3.
4. The rabbit monoclonal antibody according to claim 2, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID No.7, or comprises an amino acid sequence with more than 90% homology obtained by substitution, deletion and/or addition of one or more amino acids and/or terminal modification of any one or more amino acids of the amino acid sequence shown in SEQ ID No. 7.
5. An isolated polynucleotide encoding the rabbit monoclonal antibody of any one of claims 1-4.
6. A recombinant expression vector comprising the polynucleotide of claim 5.
7. Use of a rabbit monoclonal antibody according to any one of claims 1-4 for the preparation of an immunohistochemical detection kit for labeling human NUT proteins in normal and diseased tissue.
8. The use of claim 7, wherein the normal tissue comprises testis, tonsil, thyroid, pancreas, and the diseased tissue comprises tumor tissue comprising midline carcinoma tumor cells and papillary thyroid carcinoma tumor cells.
9. An immunohistochemical detection kit for human NUT protein, characterized in that the immunohistochemical detection kit comprises a primary antibody containing the monoclonal antibody of any one of claims 1 to 4.
10. The immunohistochemical detection kit according to claim 9, wherein the kit further comprises an antigen retrieval solution, an endogenous peroxidase blocking agent, a hypersensitive secondary antibody reagent, a DAB substrate buffer solution, a DAB concentrated color developing solution, a hematoxylin staining solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311712149.8A CN117659187B (en) | 2023-12-13 | 2023-12-13 | Anti-human NUT protein rabbit monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311712149.8A CN117659187B (en) | 2023-12-13 | 2023-12-13 | Anti-human NUT protein rabbit monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117659187A true CN117659187A (en) | 2024-03-08 |
| CN117659187B CN117659187B (en) | 2024-05-14 |
Family
ID=90078695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311712149.8A Active CN117659187B (en) | 2023-12-13 | 2023-12-13 | Anti-human NUT protein rabbit monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117659187B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110213012A1 (en) * | 2008-07-23 | 2011-09-01 | The Brigham And Women's Hospital, Inc. | Treatment of Cancers Characterized by Chromosomal Rearrangement of the NUT Gene |
| CN116716399A (en) * | 2022-10-18 | 2023-09-08 | 南京世和基因生物技术股份有限公司 | Detection method, kit and probe library of NUTM1 fusion gene |
-
2023
- 2023-12-13 CN CN202311712149.8A patent/CN117659187B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110213012A1 (en) * | 2008-07-23 | 2011-09-01 | The Brigham And Women's Hospital, Inc. | Treatment of Cancers Characterized by Chromosomal Rearrangement of the NUT Gene |
| CN116716399A (en) * | 2022-10-18 | 2023-09-08 | 南京世和基因生物技术股份有限公司 | Detection method, kit and probe library of NUTM1 fusion gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117659187B (en) | 2024-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chang et al. | Dystrophin in electric organ of Torpedo californica homologous to that in human muscle | |
| CN114989303B (en) | anti-CD 56 recombinant rabbit monoclonal antibody and application thereof | |
| CN101929999B (en) | Kit for detecting anti-moesin antibody | |
| WO2014052672A1 (en) | Anti-uroplakin ii antibodies systems and methods | |
| CA1339095C (en) | Purification of cancer-associated protein and preparation of antibody thereto | |
| CN107001437A (en) | The inspection method and inspection medicine of ovary clear cell adenocarcinoma | |
| CN108795880A (en) | Generate people's thymidine kinase 1（TK1）The mouse hybridoma cell strain of monoclonal antibody specific and its application | |
| JP3259768B2 (en) | Testing methods for kidney disease | |
| AU653536B2 (en) | Measuring non-dystrophin proteins and diagnosing muscular dystrophy | |
| CN115667304A (en) | Anti-human LAG-3 antibodies and their use in Immunohistochemistry (IHC) | |
| CN117659187B (en) | Anti-human NUT protein rabbit monoclonal antibody and application thereof | |
| CN115044560B (en) | Hybridoma cell strain, anti-human ERG protein monoclonal antibody and application thereof | |
| JP2726264B2 (en) | Assays and antibodies for N-myc proteins | |
| JP6345596B2 (en) | Method for detecting primary aldosteronism and monoclonal antibody | |
| CN116554319A (en) | Preparation and application of anti-human integrase interaction molecule 1 (INI 1) rabbit monoclonal antibody | |
| CN118063605A (en) | Anti-human BRG1 rabbit monoclonal antibody, preparation method and application thereof | |
| EP0908466B1 (en) | Anti-human lect2 antibody, cells producing the same, and method and kit for assaying the same | |
| CN114990074B (en) | Hybridoma cell strain, anti-human fumarate hydratase monoclonal antibody and application thereof | |
| CN117362438B (en) | RELT-resistant recombinant monoclonal antibody, and preparation method and application thereof | |
| CN104945496A (en) | Polypeptide and application thereof in preparing and purifying EHD2-specific antibody | |
| CN115724966A (en) | Anti-human pairing box gene 2 rabbit monoclonal antibody, preparation method and application thereof | |
| CN114507286B (en) | anti-PD-L1 recombinant rabbit monoclonal antibody and application thereof | |
| EP1876447A1 (en) | Diagnostic agent for tumor | |
| CN116554333A (en) | Anti-human mucin 6 (MUC 6) rabbit monoclonal antibodies and uses thereof | |
| TWI702400B (en) | Method for detecting ovarian cancer, method for monitoring progression of ovarian cancer in an ovarian cancer patient, and use of a reagent that recognizes a cleaved c3 polypeptide |
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 |