CN117384291A - Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell - Google Patents
Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell Download PDFInfo
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- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3007—Carcino-embryonic Antigens
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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- C07K2317/565—Complementarity determining region [CDR]
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- G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
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Abstract
The invention relates to the technical field of genetic engineering, and discloses an anti-human SALL4 rabbit monoclonal antibody, a preparation method and application thereof, a polynucleotide molecule, an expression vector and a host cell, wherein the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises 3 heavy chain complementarity determining regions, and the amino acid sequences of the heavy chain complementarity determining regions are shown in SEQ ID NO. 8-10; the light chain variable region comprises 3 light chain complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 3-5. The monoclonal antibody has high specificity, can specifically identify cells containing the human SALL4 protein, and is suitable for immunological detection, especially immunohistochemical detection.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-human SALL4 rabbit monoclonal antibody, a preparation method and application thereof, a polynucleotide molecule, an expression vector and a host cell.
Background
SALL gene family members can encode putative zinc finger transcription factors that are highly expressed during development. SALL4 and other totipotency-modulating molecules such as Oct-4 and Nanog begin to express early in development. Recent studies have shown that SALL4 is a major regulator molecule that regulates its own expression and Oct-4 expression in a transcriptional regulatory feedback loop that controls stem cell totipotency and stem cell fate. Immunohistochemical studies showed that SALL4 is a sensitive and specific diagnostic marker for primary germ cell tumors and yolk sac tumors. Studies have shown that SALL4 is expressed structurally in Acute Myelogenous Leukemia (AML) cells and may be an effector of the Wnt/β -catenin signaling pathway in disease cells. In addition, SALL4 mutations are associated with human malformation syndrome such as Duane radius line syndrome (Okihiro syndrome) and apical kidney-eye syndrome.
The SALL4 gene is responsible for regulating self-renewal of embryonic stem cells and is a key gene in many tumors. SALL4 is a carcinoembryonic protein, and studies have shown that SALL4 is a sensitive and specific marker for seminoma and ovarian primordial germ cell tumors, and is also a progenitor cell subset marker for invasive phenotypic hepatocellular carcinoma.
Disclosure of Invention
The invention provides an anti-human SALL4 rabbit monoclonal antibody, a preparation method and application thereof, a polynucleotide molecule, an expression vector and a host cell. The monoclonal antibody has high specificity, can specifically identify cells containing the human SALL4 protein, and is suitable for immunological detection, especially immunohistochemical detection.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides an anti-human SALL4 rabbit monoclonal antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region;
the heavy chain variable region comprises 3 heavy chain complementarity determining regions, and the amino acid sequence of the heavy chain complementarity determining regions is shown in SEQ ID NO. 8-10;
the light chain variable region comprises 3 light chain complementarity determining regions, and the amino acid sequence of the light chain complementarity determining regions is shown in SEQ ID NO. 3-5.
In the above technical scheme, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
In the technical scheme, the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the amino acid sequence of the light chain is shown as SEQ ID NO. 1.
The invention also provides a polynucleotide molecule which codes for the monoclonal antibody.
The invention also provides an expression vector comprising the polynucleotide molecule.
The invention also provides a host cell transformed with the expression vector.
The invention also provides a preparation method of the anti-human SALL4 rabbit monoclonal antibody, which comprises the following steps: immunizing rabbits by adopting 954 th to 1053 rd amino acid fragments of the C end of human SALL4 protein as immunogens, separating single antigen-specific B lymphocyte culture from spleen cells, extracting gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the monoclonal antibody through specific primers, constructing an expression vector, transfecting host cells to obtain a supernatant containing the monoclonal antibody, and purifying to obtain the monoclonal antibody, wherein the sequences of the 954 th to 1053 rd amino acid fragments of the C end of the human SALL4 protein are shown as SEQ ID NO. 11.
The invention also provides application of the monoclonal antibody in preparing a human SALL4 protein detection reagent and/or a detection kit, wherein the detection method comprises at least one of an enzyme immunoassay method, an enzyme-linked immunosorbent assay, an immunohistochemical method, an immunofluorescence method, an immunoblotting method and a flow cytometry method.
In the above technical solution, the detection method is preferably an immunohistochemical method.
The invention has the beneficial effects that: the invention selects 954 th to 1053 rd amino acid fragments of the C end of the human SALL4 protein as antigens, and the gene fragments expressed by escherichia coli Rosetta, the protein is expressed in the supernatant, finally the human SALL4 recombinant protein with higher purity is obtained. Human SALL4 recombinant protein is used as immunogen to immunize New Zealand white rabbits, and monoclonal antibody development technology based on single B lymphocyte screening and culture is used to obtain rabbit monoclonal antibodies resisting SALL4 protein and heavy chain and light chain sequences. The anti-human SALL4 rabbit monoclonal antibody prepared by the invention has high specificity, can specifically identify cells containing human SALL4 protein, and is suitable for immunological detection, in particular immunohistochemical detection.
Drawings
FIG. 1 is a diagram of purification of human SALL4 protein;
FIG. 2 is a schematic diagram of construction of an expression vector containing a heavy chain constant region of a rabbit monoclonal antibody;
FIG. 3 is a schematic diagram of construction of an expression vector containing a rabbit monoclonal antibody light chain constant region;
FIG. 4 shows the staining behavior of anti-human SALL4 rabbit monoclonal antibodies in human seminomas.
Detailed Description
The invention will be further described with reference to specific examples for better illustrating the objects, technical solutions and advantages of the invention. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.
The invention provides an anti-human SALL4 rabbit monoclonal antibody, which has the amino acid sequence as follows:
amino acid sequence of the light chain variable region:
AQALTQTPSSVSAAVGGTVTISCQSSQNVYDANALSWYQQKPGQPPKRLIYSASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAASYYCAGVYYGKIGFGGGTEVVVK(SEQ ID NO.2)
amino acid sequence of heavy chain variable region:
QSLEESGGRLVTPGGSLTLTCTVSGFSLSSNSITWVRQAPGKGLEWIGTMNIDNSTYYMSWAKGRLTISKTSTTVTLKMTSLTTEDTATYFCARIAVVSNTDIWGPGTLVTVSF(SEQ ID NO.7)
amino acid sequence of CDR1 of the light chain complementarity determining region:
QNVYDANALSW(SEQ ID NO.3)
amino acid sequence of CDR2 of the light chain complementarity determining region:
LIYSASTLASGV(SEQ ID NO.4)
amino acid sequence of CDR3 of the light chain complementarity determining region:
AGVYYGKIGF(SEQ ID NO.5)
amino acid sequence of CDR1 of the heavy chain complementarity determining region:
FSLSSNSIT(SEQ ID NO.8)
amino acid sequence of CDR2 of the heavy chain complementarity determining region:
WIGTMNIDNSTYYMSWAK(SEQ ID NO.9)
amino acid sequence of CDR3 of the heavy chain complementarity determining region:
YFCARIAVVSNTDI(SEQ ID NO.10)
amino acid sequence of light chain:
MDTRAPTQLLGLLLLWLPGARCAQALTQTPSSVSAAVGGTVTISCQSSQNVYDANALSWYQQKPGQPPKRLIYSASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAASYYCAGVYYGKIGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.1)
amino acid sequence of heavy chain:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGGSLTLTCTVSGFSLSSNSITWVRQAPGKGLEWIGTMNIDNSTYYMSWAKGRLTISKTSTTVTLKMTSLTTEDTATYFCARIAVVSNTDIWGPGTLVTVSFGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6)
the invention also provides a preparation method of the anti-human SALL4 rabbit monoclonal antibody, which comprises the following steps: the method comprises the steps of taking 954 th to 1053 rd amino acid fragments (SEQ ID NO. 11) of the C end of human SALL4 protein as an immunogen, immunizing rabbits, separating single antigen-specific B lymphocyte culture from spleen cells, extracting gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the monoclonal antibody through specific primers, constructing an expression vector, transfecting host cells, obtaining a supernatant containing the monoclonal antibody, and purifying to obtain the anti-human SALL4 rabbit monoclonal antibody.
The preparation and application of the above anti-human SALL4 rabbit monoclonal antibodies are illustrated below.
Test example 1 expression purification of recombinant SALL4 protein
1. Construction of protein expression plasmid
The corresponding nucleic acid fragments from 954 th to 1053 rd of the C end of the human SALL4 protein are obtained by gene synthesis.
2. Protein expression
pET-32a plasmid containing SALL4 954-1053aa sequence was transformed into E.coli Rosetta strain and incubated overnight at 37℃on LB agar plates (containing 100. Mu.g/mL ampicillin) to obtain several single colony transformants.
Single colony transformants were inoculated into 2mL of LB medium (containing 100. Mu.g/mL ampicillin) in 10mL polypropylene tubes and cultured at 220rpm at 37℃for 3-4 hours, OD 600nm About 0.4 to 0.6, and then 2mL of the culture of each strain was transferred to 400mL of LB expression medium in a 1L flask, and further cultured at 220rpm at 37℃for 3 to 4 hours. When OD is 600nm When the bacterial liquid reaches about 0.45-0.55, 0.8mM IPTG is added, induction is carried out for 3-4 hours at 37 ℃, the induced bacterial liquid is transferred into a dry 500mL centrifugal bottle, the electronic scale is used for balancing, pure water is added when the mass is different, the centrifugal bottle is centrifuged at 4000rpm for 10 minutes, the supernatant is discarded, the centrifugal bottle is placed in the normal position, and the bacterial liquid is stored in a freezer at minus 20 ℃.
30mL of the bacterial suspension (50 mM Tris-300mM NaCl) was used to suspend the cells in a centrifuge bottle. The suspended bacterial liquid was transferred to a 50mL round bottom centrifuge tube, placed in an ice box and fixed with ice. Selecting an amplitude transformer, placing the amplitude transformer into a bacteria breaking cabin, wherein the power is 350W, the bacteria breaking time is 3s, the interval time is 3s, the time is counted down for 5min, then placing the amplitude transformer into an ice-water mixture for cooling for 5min, and repeating the steps for breaking bacteria for 5min. After the completion of the sterilization, the mixture was centrifuged at 9000rpm for 10min to obtain a supernatant (2) and a precipitate.
Crushing for the second time: 30mL of a bacteria-destroying solution (2M urea-PBS buffer solution) is taken and poured into a sediment, the sediment is uniformly blown, the mixture is transferred into a 50mL round bottom centrifuge tube, the power is 350W, the bacteria-destroying time is 3s, the interval time is 3s, the bacteria-destroying time is 0-5min (the bacteria solution is determined according to the sediment amount and the texture, and the bacteria solution is placed in ice cubes), the mixture is centrifuged at 9000rpm for 10min to obtain a supernatant and inclusion bodies, wherein a lane 1 is the supernatant (2), a lane 8 is the inclusion bodies, and a lane 6 and a lane 7 are proteins eluted by 300mM and 500mM imidazole (as shown in FIG. 1). And finally obtaining the corresponding protein fragment.
Wherein, the sequence of the amino acid fragments from 954 th to 1053 rd of the human SALL4 protein is as follows:
PKEILAPSVNVDPVVWNQYTSMLNGGLAVKTNEISVIQSGGVPTLPVSLG ATSVVNNATVSKMDGSQSGISADVEKPSATDGVPKHQFPHFLEENKIAVS(SEQ ID NO.11)
3. protein acquisition
The SALL4 protein was purified by affinity chromatography from supernatant (2) using Ni-IDA affinity purification matrix, and the purification results are shown in FIG. 1. From left to right, lane 1 in FIG. 1 is supernatant (2) (abbreviated as "upper 2"), lanes 2 and 3 are BSA at 0.4mg/mL and 0.2mg/mL, respectively, lane 4 is marker, lane 5 is flow-through during purification (abbreviated as "FT"), and lanes 6 and 7 are 2-fold dilutions of SALL4 protein collected (abbreviated as "x 2"). From the SDS-PAGE gel diagram of FIG. 1, it can be discerned that: a recombinant SALL4 protein with a purity of 95% was obtained, comprising a SALL4 protein fragment, a His tag and a Ni-IDA protein tag.
Test example 2 preparation and purification method of rabbit monoclonal antibody
1. Immunization of animals
3 novel cells were immunized with recombinant human SALL4 protein (purified from test 1)A white rabbit; immunization of each white rabbit with 300 μg of immunogen, mixing the immunogen with an equivalent amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifier before the first immunization, and subcutaneously injecting the emulsifier into the abdomen and back of the rabbits at multiple points; 150 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) at 2 weeks intervals after the first immunization to prepare an emulsifier, which was injected subcutaneously in the abdomen and back of rabbits in multiple spots to boost the immunization twice. Three immunizations were followed by collection of rabbit serum samples, ELISA for human SALL4 protein titers, dilution of serum at 1:243K, ELISA for titers, OD 450nm Rabbits exceeding 0.1 were boosted subcutaneously with 300 μg immunogen at multiple points and spleens were taken three days later.
2. Isolation of spleen cells
Taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by rolling the tail end of the pressed part. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company), gently blowing off cell clusters by using a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
3. B lymphocyte sorting
The method described in the specification of Chinese patent CN201910125091.4 (patent name: method for efficiently isolating single antigen-specific B lymphocytes from spleen cells) was adopted.
4. Identification of positive clones by antigen-coated ELISA of cultured B cell supernatants
Cells of positive clones were collected and lysed, and RNA was extracted using the Quick-RNATM MicroPrep kit (available from ZYMO Co.) and reverse transcribed into cDNA.
The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of a naturally paired rabbit monoclonal antibody from the cDNA of the corresponding positive clone, and a plurality of clones are selected for sequencing, and the sequencing work is completed by Jin Kairui biotechnology limited company.
Wherein, the PCR reaction system is as follows: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2 XGloria HiFi (supplied by Wuhan Aibotai Biotechnology Co., ltd.) and 6.5. Mu.L of N.F H2O.
Wherein, the light chain variable region primer pair is:
5’-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3’
5’-cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG-3’
the heavy chain variable region primer pair is:
5’-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3’
5’-gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG-3’
PCR amplification procedure: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.
5. Monoclonal antibody preparation and purification
(1) And (3) loading the heavy chain genes and the light chain genes of the plurality of rabbit monoclonal antibodies selected in the step (4) on the expression vectors respectively, wherein the used mammal expression vector pBR322 is shown in figures 2 and 3. In FIGS. 2 and 3, pBR322 origin and f1 origin are replication promoters in E.coli (E.Coli), ampcilin is a plasmid resistance gene, CMV immearly promotor is a promoter in eukaryotes, SV40 PA terminator is a tailing signal, heavy chain constant in FIG. 2 is a nucleotide sequence of a heavy chain constant region of a rabbit monoclonal antibody, and Light chain constant in FIG. 3 is a nucleotide sequence of a light chain constant region of a rabbit monoclonal antibody.
(2) Mammalian cell (e.g., CHO, HEK293, etc.) expression vectors containing the heavy chain constant region (FIG. 2) and the light chain constant region (FIG. 3) of the rabbit monoclonal antibodies were routinely linearized with NheI and XbaI restriction enzymes, respectively.
(3) Purifying the PCR product amplified in the step 4, and respectively constructing a heavy chain variable region gene and a light chain variable region gene into corresponding mammal expression vectors by adopting a homologous recombination mode; after sequencing verification, the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies are transfected into 293F cells together; at a 293F cell viability of 75% + -5%, a recombinant rabbit monoclonal antibody recognizing human SALL4 was obtained in the culture supernatant after 72-96 hours of transfection.
Purifying recombinant rabbit monoclonal antibody recognizing human SALL4 protein from transfected culture medium supernatant by using protein A affinity gel resin, verifying antibody purity by SDS-PAGE gel electrophoresis, subpackaging after verification, and preserving at-20deg.C for use. The sequences of the selected rabbit monoclonal antibodies were as described above.
Test example 3 specificity identification
Immunohistochemical tissue chip staining and identification
1. Chip selection:
human SALL4 protein is expressed in almost all primordial germ cell tumors, and may be partially expressed in the primordial neuroepithelium of immature teratomas. Thus, seminoma, embryonal carcinoma tissue was selected as positive samples, liver cancer, tonsil, anaplastic Large Cell Lymphoma (ALCL), hodgkin's lymphoma kidney, liver, thymoma tissue as negative controls.
2. IHC staining and analysis:
sample preparation, baking of the slices: placing paraffin slices on a slice rack in the same direction, and placing the paraffin slices into a constant temperature box at 56 ℃ to bake the slices for 30min; simultaneously placing the dewaxing liquid 1 cylinders into an incubator at 56 ℃; dewaxing to water: placing paraffin slices and a slice frame into a dewaxing liquid 1 jar, taking out the paraffin slices and the slice frame from an incubator and placing the paraffin slices and the slice frame into a normal temperature dewaxing liquid 2 jar, taking out the paraffin slices and immersing the paraffin slices into the dewaxing liquid 2 jar, and sequentially placing paraffin slices into the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 1, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3 according to the sequence of the dewaxing liquid 2, the dewaxing liquid reagent jar for 5min each, and the absolute ethyl alcohol reagent jar for 3min each; the sections were washed with running water for 3min.
Antigen retrieval: 0.01M Tris-EDTA repair solution (pH 9.0) was autoclaved.
Inactivation of endogenous peroxidases: immersing and washing for 3 times by using PBS buffer solution for 1min each time, and removing the buffer solution on the slice; the sections were completely immersed in 3% hydrogen peroxide solution and incubated at room temperature for 10min.
Closing: immersing and washing for 3 times by using PBS buffer solution for 3min each time, and removing the buffer solution on the slice; an immunohistochemical water pen is used for delineating a tissue region to be detected on a slide, and a blocking liquid-PBS blocking liquid is dripped into the delineating region; the sections were placed horizontally in an incubation wet box with water at the bottom, incubated at room temperature for 30min, and counted from the addition of the blocking solution.
Incubation resistance: removing the sealing liquid, dripping a primary antibody diluted by an antibody diluent-PBS working liquid on the tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 60min at normal temperature; removing antibody working solution, quickly rinsing with PBS buffer solution for 1 time, soaking and washing with the buffer solution PBS for 3 times, and 3 minutes each time; the soaking and washing period needs to be repeatedly lifted up and down for a plurality of times (the primary anti-dilution ratio is 1:100).
Secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (agent bottle A) on a tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 25min at normal temperature; removing reagents on the sections, quickly rinsing the sections for 1 time by using buffer PBS, soaking and washing the sections for 3 times by using the buffer PBS for 3 minutes each time; the soaking and washing period needs to be repeatedly lifted up and down for a plurality of times.
Color development: dropwise adding a color development liquid working solution on a tissue slice, closely observing the color change condition under a microscope, and obtaining proper dyeing intensity; immersing the slices in a large amount of distilled water to terminate the color development; after the development was terminated, the sections were washed in running water for 10 minutes.
Counterstaining: the slightly drained sections were counterstained in Mayer's hematoxylin for 1min, after counterstaining was completed, washed with running water for 3min.
Returning blue: the slightly drained slices were immersed in a saturated aqueous solution of lithium carbonate for bluing for 3s and washed with running water for 3min.
Dehydrating: soaking the cleaned slice in absolute ethyl alcohol for 1 time, lifting up and down for several times during the soaking period, and taking out after timing for 10 seconds; and (3) placing the mixture in a constant-temperature blast drying oven to be completely dried at a high temperature (54-58 ℃).
Sealing piece: and (3) dripping a proper amount of neutral gum into the center of the slice, covering a cover glass, wherein the proper amount of gum is needed, and the cover glass is needed to cover tissues completely after the cover glass is added, so that no gum overflows.
Slice scanning.
The anti-human SALL4 rabbit monoclonal antibody prepared using test example 2 was specifically colored in human seminoma.
Fig. 4 shows the staining behavior in human seminomas. The result shows that the anti-human SALL4 rabbit monoclonal antibody prepared in test example 2 has accurate staining and positioning, clear staining, no nonspecific staining and clean background.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. An anti-human SALL4 rabbit monoclonal antibody, characterized in that: the monoclonal antibodies comprise a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region;
the heavy chain variable region comprises 3 heavy chain complementarity determining regions, and the amino acid sequence of the heavy chain complementarity determining regions is shown in SEQ ID NO. 8-10;
the light chain variable region comprises 3 light chain complementarity determining regions, and the amino acid sequence of the light chain complementarity determining regions is shown in SEQ ID NO. 3-5.
2. The monoclonal antibody of claim 1, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
3. The monoclonal antibody of claim 1, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the amino acid sequence of the light chain is shown as SEQ ID NO. 1.
4. A polynucleotide molecule characterized by: the polynucleotide molecule encodes the monoclonal antibody of any one of claims 1-3.
5. An expression vector, characterized in that; the expression vector comprises the polynucleotide molecule of claim 4.
6. A host cell, characterized in that: the host cell is transformed with the expression vector of claim 5.
7. The method for producing a monoclonal antibody according to claim 1, characterized in that: the method comprises the following steps: immunizing rabbits by adopting 954 th to 1053 rd amino acid fragments of the C end of human SALL4 protein as immunogens, separating single antigen-specific B lymphocyte culture from spleen cells, extracting gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the monoclonal antibody through specific primers, constructing an expression vector, transfecting host cells to obtain a supernatant containing the monoclonal antibody, and purifying to obtain the monoclonal antibody, wherein the sequences of the 954 th to 1053 rd amino acid fragments of the C end of the human SALL4 protein are shown as SEQ ID NO. 11.
8. Use of a monoclonal antibody according to any one of claims 1-3 for the preparation of a human SALL4 protein assay reagent and/or assay kit, characterized in that: the detection method comprises at least one of enzyme immunoassay, enzyme-linked immunosorbent assay, immunohistochemical method, immunofluorescence method, immunoblotting method and flow cytometry.
9. The use according to claim 8, characterized in that: the detection method is an immunohistochemical method.
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