CN116082512B

CN116082512B - Monoclonal antibody for CDKn1A_p21CIP1 protein, and preparation method and application thereof

Info

Publication number: CN116082512B
Application number: CN202211625993.2A
Authority: CN
Inventors: 代海艳; 胡文娟; 孙倩; 李倩倩; 程瑶; 张云; 李博; 吴海
Original assignee: Wuhan Abclonal Inc
Current assignee: Wuhan Abclonal Inc
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2023-10-13
Anticipated expiration: 2042-12-15
Also published as: CN116082512A

Abstract

The invention provides a monoclonal antibody aiming at human CDKN1A_p21CIP1 protein, a preparation method and application thereof. The sequence of the light chain complementarity determining region of the rabbit monoclonal antibody aiming at the human CDKn1A_p21CIP1 protein is shown as SEQ ID NO. 3-SEQ ID NO. 5, the sequence of the heavy chain complementarity determining region is shown as SEQ ID NO. 8-SEQ ID NO. 10, and the rabbit monoclonal antibody can specifically identify cells of the human CDKn1A_p21CIP1 protein, is suitable for immunological detection, particularly pathological-level immunohistochemical detection, and can be used for disease diagnosis.

Description

Monoclonal antibody for CDKn1A_p21CIP1 protein, and preparation method and application thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a monoclonal antibody aiming at CDKn1A_p21CIP1 protein, a preparation method and application thereof.

Background

P21 (CDKN 1A, also designated as P21WAF1/CIP1/SDI 1) is a cyclin-dependent kinase (CDK) inhibitor encoded by the CDKN1A gene, located on the sixth chromosome short arm downstream of the P53 gene, and is an important member of the CIP/KIP family. The p21 protein is rich in arginine because of its protein relative molecular mass of 21kDa and consists of 164 amino acids. The p21 protein exists in the form of a tetrameric complex in normal cells, including cell Cycle Dependent Kinases (CDKs), cyclin (Cyc-lin), and Proliferating Cell Nuclear Antigen (PCNA). The P21 protein inhibits CDK and PCNA, while P53 or cell mitogen stimulates induction of P21 transcription, disrupting the phosphorylation of RB protein, and cell cycle arrest in G1 until damaged DNA is repaired.

The p21 protein is involved in cell cycle arrest, DNA replication and repair, cell proliferation and differentiation, senescence and apoptosis, and is closely related to tumor development. p21 can exert not only anticancer effects but also carcinogenesis depending on cell type, cell background, subcellular localization and post-translational modification. Abnormal expression of p21 protein affects the regulation of Cyclin, CDK and kinase activity thereof, and thus affects cell proliferation and differentiation, ultimately leading to tumor development. The p21 protein can be used as a reference index for judging the differentiation degree and prognosis of tumors, can be used as a diagnosis marker for malignant tumors such as colon cancer and the like, and can be used as a treatment target for inhibiting tumor growth.

Therefore, development of a detection technology for cdkn1a_p21cip1 protein is needed.

Disclosure of Invention

Based on the above, it is necessary to provide a rabbit monoclonal antibody against CDKn1A_p21CIP1 protein, and a preparation method and application thereof, and to realize immunological detection of cellular p21CIP1 protein by utilizing high specificity of the rabbit monoclonal antibody.

The invention adopts the following technical scheme:

the invention provides a rabbit monoclonal antibody aiming at human CDKn1A_p21CIP1 protein, wherein the sequences of light chain complementarity determining regions are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, the sequence of a light chain variable region is shown as SEQ ID NO. 2, and the full-length sequence of a light chain is shown as SEQ ID NO. 1; the sequences of the heavy chain complementarity determining regions are shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, the sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the full-length sequence of the heavy chain is shown as SEQ ID NO. 6.

The invention also provides a recombinant protein for preparing the rabbit monoclonal antibody aiming at the human CDKn1A_p21CIP1 protein, wherein the recombinant protein comprises an amino acid fragment with a sequence shown as SEQ ID NO. 11. Preferably, the recombinant protein is recombinantly expressed by E.coli, comprising a CDKn1A_p21CIP1 protein fragment, a glutathione thiol transferase (GST) tag, and a histidine protein tag.

The present invention can provide a nucleotide sequence encoding the above-described rabbit monoclonal antibody against human cdkn1a_p21cip1 protein, an expression vector or host comprising the above-described nucleotide sequence.

The invention also provides a reagent for preparing and detecting the human CDKn1A_p21CIP1 protein by using the rabbit monoclonal antibody aiming at the human CDKn1A_p21CIP1 protein. The detection method is immunodetection, and can be at least one of immunohistochemistry, immunoblotting and immunoprecipitation.

The invention also provides a preparation method of the rabbit monoclonal antibody aiming at the human CDKN1A_p21CIP1 protein, which comprises the following steps: respectively loading genes of rabbit monoclonal antibodies aiming at human CDKN1A_p21CIP1 proteins into expression vectors, and transfecting hosts; culturing to obtain a supernatant containing the rabbit monoclonal antibody; purifying to obtain the final product.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, a specific amino acid fragment at the C end of CDKn1A_p21CIP1 protein is selected as an antigen, escherichia coli Rosetta is expressed to obtain recombinant protein as an immunogen, new Zealand white rabbits are immunized, and then a monoclonal antibody development technology for resisting the CDKn1A_p21CIP1 protein is obtained based on single B lymphocyte screening and culturing, so that cells capable of specifically recognizing the human CDKn1A_p21CIP1 protein are verified, and the method is suitable for immunological detection, in particular immunohistochemical detection.

Drawings

FIG. 1 is a gel diagram of SDS-PAGE for detection of CDKn1A_p21CIP1 protein expression in inclusion bodies in example 1.

FIG. 2 is a gel diagram showing the result of GST tag purification of CDKn1A_p21CIP1 protein detected by SDS-PAGE in example 1.

FIG. 3 is a schematic diagram of the construction of an expression vector containing the heavy chain constant region of a rabbit monoclonal antibody in example 2.

FIG. 4 is a schematic diagram of the construction of an expression vector comprising the light chain constant region of a rabbit monoclonal antibody according to example 2.

FIG. 5 is a SDS-PAGE gel of the rabbit monoclonal antibodies purified in example 2.

FIG. 6 is a diagram showing detection of recognition specificity of rabbit monoclonal antibodies against CDKn1A_p21CIP1 in cell samples by immunoblotting method of example 3.

FIG. 7 is a staining localization map of CDKn1A_p21CIP1 in human esophageal cancer tissue samples using immunohistochemistry according to example 4.

Detailed Description

The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

The technical concept of the invention is to provide a rabbit monoclonal antibody aiming at CDKn1A_p21CIP1 protein, and a preparation method and application thereof. The sequence of the rabbit monoclonal antibody against the cdkn1a_p21cip1 protein was:

light chain amino acid sequence:

MDTRAPTQLLGLLLLWLPGATFAAVLTQTPSPVSAAVGGTVTISCQSSHTIYNKYDLTWYQQKPGQPPKVLIYRASRLASGVPSRFSGSGSGTQFTLTISGVQCDDSATYYCLAGYDDDGAAEGFGGGTEVVVEGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.1)。

amino acid sequence of the light chain variable region:

MDTRAPTQLLGLLLLWLPGATFAAVLTQTPSPVSAAVGGTVTISCQSSHTIYNKYD LTWYQQKPGQPPKVLIYRASRLASGVPSRFSGSGSGTQFTLTISGVQCDDSATYYCLAG YDDDGAAEGFGGGTEVVVE(SEQ ID NO.2)。

amino acid sequence of CDR1 of the light chain complementarity determining region:

HTIYNKYDLTW(SEQ ID NO.3)。

amino acid sequence of CDR2 of the light chain complementarity determining region:

LIYRASRLASGV(SEQ ID NO.4)。

amino acid sequence of CDR3 of the light chain complementarity determining region:

LAGYDDDGAAEGF(SEQ ID NO.5)。

heavy chain amino acid sequence:

METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCSVSGFSLSSFAMTWVRQVPGKGLEWIGIIGTSGRISSASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCARCEYYSDGTDDTDGFYFNIWGPGTLVTVSAGTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6)。

amino acid sequence of heavy chain variable region:

QSVEESGGRLVTPGTPLTLTCSVSGFSLSSFAMTWVRQVPGKGLEWIGIIGTSGRISSASWAKGR FTISKTSTTVDLKMTSPTTEDTATYFCARCEYYSDGTDDTDGFYFNIWGPGTLVTVSAGTVSS(SEQ ID NO.7)。

amino acid sequence of CDR1 of the heavy chain complementarity determining region:

FSLSSFAMT(SEQ ID NO.8)。

amino acid sequence of CDR2 of the heavy chain complementarity determining region:

WIGIIGTSGRISSASWAK(SEQ ID NO.9)。

amino acid sequence of CDR3 of the heavy chain complementarity determining region:

YFCARCEYYSDGTDDTDGFYFNI(SEQ ID NO.10)。

the invention is directed to a rabbit monoclonal antibody to human CDKn1A_p21CIP1 protein, wherein the light chain constant region is a kappa chain and the heavy chain constant region is of the IgG1 type.

According to the invention, the recombinant protein is utilized to immunize New Zealand white rabbits, and a monoclonal antibody development technology based on single B lymphocyte screening and culture is utilized to obtain the rabbit monoclonal antibody resisting the human CDKn1A_p21CIP1 protein, and the heavy chain and light chain sequences, so that cells of the human CDKn1A_p21CIP1 protein can be specifically identified, and the method is suitable for immunological detection, in particular immunohistochemical detection.

The following is illustrative:

example 1

The embodiment provides a preparation method of a recombinant protein immunogen, which comprises the following steps:

1.1 construction of protein expression plasmids

Using the sequence synthesized by the gene corresponding to the 1 st to 164aa of human CDKn1A_p21CIP1 as a cloning template, amplifying the fragments of 1 st to 164 th target of the full length of the human CDKn1A_p21CIP1 protein by a pair of specific primers, wherein the PCR reaction system is as follows: 1. Mu.L of template, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 25. Mu.L of 2 XGloria Hi-FiPCRMasterMixWithGCBuffer (Bothack Biotechnology Co., ltd.) 22. Mu.L N.F H ₂ O。

Wherein the amino acid fragment sequences from 1 st to 164 th of the human CDKN1A_p21CIP1 protein are as follows:

MSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP(SEQ ID NO.11)。

the primer pair sequences are respectively as follows:

CDKN1A_p21CIP1-Primer-F：

5'-GGTTCCGCGTGGATCCCCGGAA ATGTCAGAACCGGCTGGG-3'(SEQ ID NO:12)；

CDKN1A_p21CIP1-Primer-R：

5'-TGGTGATGATGATGCGGCCGCTCGGGCTTCCTCTTGGAGAAGAT-3'(SEQ ID NO:13)。

PCR amplification procedure: the reaction solution is subjected to pre-denaturation at 98 ℃ for 30s, then 30 times of circulation are carried out according to the conditions of 10s at 98 ℃, 30s at 64 ℃ and 30s at 72 ℃, and finally the reaction solution is kept at 72 ℃ for 5min, and the obtained reaction solution is kept at 4 ℃.

After the amplified PCR product is purified, the PCR product is loaded on an expression vector in a homologous recombination mode, the prokaryotic expression vector is pGEX-4T-AB1 (the commercial pGEX-4T-1 vector is obtained by adding an 8 XHis tag at the C end), the plasmid is preserved after sequencing verification, and sequencing work is completed by Jin Kairui Biotechnology Co.

1.2 protein expression

pGEX-4T-AB1 plasmid containing CDKn1A_p21CIP1 1-164aa nucleotide 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.

Inoculating single colony transformant into 2mLLB culture solution (containing 100 mug/mL ampicillin) in 10mL polypropylene tube, culturing at 37deg.C and 220rpm for 3-4 hr until OD _600nm About 0.4 to 0.6, 2mL of the culture of each strain was transferred to 400mLLB expression medium in a 1L flask, and further cultured at 220rpm for 3 to 4 hours at 37 ℃. 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, pH 7.4) 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 (1) and a precipitate.

Crushing for the second time: weighing 30mL of bacteria breaking liquid (2M urea-PBS Buffer solution, pH value is 7.4), pouring the bacteria breaking liquid into a sediment, blowing the sediment uniformly, transferring the sediment into a 50mL round bottom centrifuge tube, wherein the power is 350W, the bacteria breaking time is 3s, the interval time is 3s, the bacteria breaking time is 0-5 min (the breaking time is determined according to the sediment amount and the texture, the bacteria liquid is placed in ice cubes), centrifuging the bacteria breaking liquid at 9000rpm for 10min to obtain supernatant (2) and inclusion bodies, and respectively diluting the obtained supernatant (1) and supernatant (2) by using Loading Buffer for 2 times, and diluting the inclusion bodies by 2 times and 10 times; the obtained samples of 2-fold diluted supernatant (1) (abbreviated as "upper 1"), 2-fold diluted supernatant (2) (abbreviated as "upper 2"), 2-fold diluted inclusion body (abbreviated as "×2"), and 10-fold diluted inclusion body (abbreviated as "×10") were subjected to SDS-PAGE gel electrophoresis, and the results are shown in fig. 1.

As can be seen from fig. 1, the cdkn1a_p21cip1 (1-164 aa) -GST recombinant protein is mainly expressed in inclusion bodies, the inclusion bodies are denatured by using 8M urea, the obtained supernatant is purified by adopting a Ni ion affinity chromatography method, and the purified protein needs to be subjected to inclusion body renaturation, which comprises the following specific steps: transferring the purified inclusion body protein into a dialysis bag, diluting to below 0.5mg/mL with 8M urea-PBS (pH 7.4), sequentially adding into 6M urea-PBS renaturation solution, 4M urea-PBS renaturation solution, 3M urea-PBS renaturation solution, 2M urea-PBS renaturation solution, 1M urea-PBS renaturation solution, 0.5M urea-PBS renaturation solution and 0M urea-PBS renaturation solution, and each gradient for 4h. Proteins renatured to 0M urea-PBS were then dialyzed 2 times, each for 2h, against PBS buffer (protein: pbs=1:100). Finally, the dialyzed protein was concentrated to the desired volume using PEG 20000.

The recombinant protein with higher purity is obtained, and comprises a CDKn1A_p21CIP1 protein fragment, a glutathione-transferase (GST) tag and a histidine protein tag, the result is shown in figure 2, and the result shows that: and (3) obtaining recombinant protein with purity of more than 85% after renaturation.

Example 2

The present example provides a method for preparing a rabbit monoclonal antibody to human CDKn1A_p21CIP1 protein, comprising the steps of:

2.1 immunization of animals: 6 New Zealand white rabbits are immunized by using the active recombinant protein prepared in the example 1 as an immunogen; each white rabbit was immunized 300. Mu.g, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant to prepare an emulsion before the first immunization, and the emulsion was subcutaneously injected at the abdomen and back of the rabbit at multiple points. After the primary immunization, 150 mug of immunogen is mixed with an equal amount of incomplete Freund's adjuvant every 2 weeks to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting. After three immunizations, serum samples of rabbits were collected, the titer against human CDKN1A_p21CIP1 protein was determined by ELISA, and after the last immunization, the serum was purified to antibodies, WB and IHC were used to detect endogenous samples, rabbits with high serum titer and best endogenous detection were obtained, and after three days, spleens were obtained by subcutaneous multipoint injection of 300. Mu.g immunogen for boosting.

2.2 isolation of spleen cells: taking out a culture dish in a sterile operation table, adding 30-40 mL 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 using the tail end of the pressed part of the grinding rod. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed 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.

2.3B lymphocyte sorting: see patent 201910125091.4, methods for efficiently isolating individual antigen-specific B lymphocytes from spleen cells.

2.4 cloning of genes encoding rabbit monoclonal antibodies:

the cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cell collection of positive clonesAfter solution, use of Quick-RNA ^TM The MicroPrep kit (available from ZYMO corporation) extracts RNA and reverse transcribes it into cDNA. The naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clone using PCR and sequenced.

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 (Botek, wuhan), 6.5. Mu.L of N.F H ₂ O。

Wherein, the light chain variable region primer pair is:

VL-Primer-F：

5'-tgaattcgagctcggtacccatggacacgagggcccccac-3'(SEQ ID NO:14)；

VL-Primer-R：

5'-cacacacacgatggtgactgttccagttgccacctgatcag-3'(SEQ ID NO:15)。

the heavy chain variable region primer pair is:

VH-Primer-F：

5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3'(SEQ ID NO:16)；

VH-Primer-R：

5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'(SEQ ID NO:17)。

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 ℃.

2.5 preparation and purification of monoclonal antibodies

2.5.1 heavy chain and light chain genes of the plurality of rabbit monoclonal antibodies selected in step 2.4 were loaded on expression vectors, respectively (see FIGS. 3 and 4). In FIGS. 3 and 4, 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. 3 is a nucleotide sequence of a heavy chain constant region of a rabbit monoclonal antibody, and Light chain constant in FIG. 4 is a nucleotide sequence of a light chain constant region of a rabbit monoclonal antibody.

The specific method comprises the following steps:

2.5.2 mammalian cell expression vectors containing the heavy chain constant region (FIG. 3) and the light chain constant region (FIG. 4) of the rabbit monoclonal antibodies were routinely linearized with NheI and XbaI restriction enzymes, respectively.

2.5.3 purifying the PCR product amplified in the step 2.4, 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; and (5) carrying out transfection for 72-96 hours and centrifuging to obtain a culture supernatant.

Purifying recombinant rabbit monoclonal antibody which recognizes human CDKn1A_p21CIP1 protein from the transfected culture medium supernatant by using protein A affinity gel resin, wherein the affinity chromatography experimental steps are as follows: the culture supernatant was transferred to a sterile 50ml centrifuge tube, centrifuged at 1000g, at 4℃or at room temperature for 10 minutes, and the supernatant was collected. The pretreated Protein A Agarose (brand: tiandi manhe; product number: SA 023100) suspension was added to the centrifuged cell supernatant and incubated at room temperature for 3-4 hours or overnight at 4 ℃. After incubation, 1000g was centrifuged for 10min, the Protein A Agarose suspension was transferred to an adsorption column, and centrifuged at room temperature for 1min with a palm centrifuge to separate the solid from the liquid and collect the flow-through. Ten times Protein A Agarose volumes of wash buffer (phosphate buffer pH 7.0) was added and the particles resuspended, centrifuged in a centrifuge, and the wash solution was collected and washed twice more. Adding elution buffer (pH of citrate buffer is 3.0) into adsorption column, centrifuging with centrifuge to obtain antibody supernatant, loading into dialysis bag (Soy Bao YA 1070), dialyzing overnight, collecting antibody, packaging after the antibody is qualified, and preserving at-20deg.C.

The SDS-PAGE running gel detection of the purified rabbit monoclonal antibody is shown in FIG. 5, and the results show that: the obtained monoclonal antibody has the advantages of correct light and heavy chain size, single strip and high antibody purity.

Example 3

The present example provides a method for the specific identification of a rabbit monoclonal antibody to human CDKn1A_p21CIP1 comprising the steps of:

cell samples HeLa (university of agricultural in China), MCF-7 (Shanghai cell bank of China), 293T (university of Wuhan) and KO sample 293T-CDKN1A-1# homozygotes (Botaike Biotechnology Co., ltd.) which do not express CDKn1A_p21CIP1 protein were taken, and the recognition specificity of the rabbit monoclonal antibody prepared in example 2 was detected by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed.

Gel protein bands were transferred to PVDF membranes in an electrotransfer system in a conventional manner. The membranes were incubated in TBST blocking solution containing 3% nonfat milk powder for 1h at room temperature, antibody CDKn1A_p21CIP1 rabbit monoclonal antibody (1:1000 dilution) was added and incubated overnight at 4 ℃. After washing the membrane with TBST, goat anti-rabbit secondary antibody diluted 1:10000 was added and incubated for 1 hour at room temperature. The membrane was washed again with TBST, ECL developing solution was added, and the result was shown in FIG. 6.

As can be seen from FIG. 6, the CDKn1A_p21CIP1 monoclonal antibody can specifically recognize MCF-7, heLa and 293T cell samples which highly express the protein, and does not recognize 293T-CDKn1A-1# homozygotes which do not express the protein KO samples.

Example 4

The present example provides a method for immunohistochemical identification of rabbit monoclonal antibody to human CDKN1A_p21CIP1 comprising the steps of:

s1, selecting a chip

Human CDKN1A_p21CIP1 protein is widely expressed in human samples, and human conventional samples such as lung, brain, colon, esophagus, placenta and tonsil and cancer samples such as colon cancer, breast cancer, esophageal cancer, liver cancer and lung cancer samples are selected for immunohistochemical detection.

S2, IHC staining and analysis

Sample preparation, baking of the slices: placing paraffin slice (Wohai Weir biotechnology Co., ltd.) on slice rack in the same direction, and baking in oven at 56 deg.C for 30min; simultaneously placing the dewaxing liquid (the original industrial and commercial Co., ltd. In Wuxi city) 1 jar together into a constant temperature box at 56 ℃; dewaxing to water: placing paraffin slices and a slice frame into a dewaxing liquid 1 jar together, taking out the paraffin slices from an incubator and placing the paraffin slices at normal temperature for 5min, taking out the paraffin slices and immersing the paraffin slices into a normal temperature dewaxing liquid 2 jar, and sequentially placing the paraffin slices into the jar according to the sequence of dewaxing liquid 2, dewaxing liquid 3, absolute ethyl alcohol 1, absolute ethyl alcohol 2 and absolute ethyl alcohol 3, wherein immersion time of each dewaxing liquid reagent jar is 5min, and immersion time of each absolute ethyl alcohol reagent jar is 3min; the sections were washed with running water for 3min.

Antigen retrieval: high pressure thermal remediation was carried out using a 0.01M sodium citrate remediation solution (pH 6.0).

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; the tissue region to be detected on the slide is defined by an immunohistochemical water pen, and a blocking solution-PBS blocking solution (5% goat serum) is dripped into the defined region; horizontally placing the slices in an incubation wet box with water at the bottom, incubating for 30min at normal temperature, and starting timing from dripping the sealing liquid;

incubation resistance: removing the blocking solution, dropwise adding a primary antibody (CDKn1A_p21CIP 1 mAb, prepared in example 2) diluted by an antibody diluent-PBS working solution onto a tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating the tissue slice at normal temperature for 60min; 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; repeatedly lifting up and down for multiple times (the primary anti-dilution ratio is 1:200) during soaking and washing;

secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (Dako REAL EnVision Detection System, peroxidase/DAB, rabbit/Mouse, HRP; dako) on a tissue slice, horizontally placing in an incubation wet box, and incubating at normal temperature for 25min; 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: immersing the slightly drained slice into Mayer's hematoxylin to counterstain the slice for 1min, and washing the slice with running water for 3min after counterstaining is completed;

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 the mixture is placed in a constant temperature blast drying box 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.

Immunohistochemical staining results were divided into: positive and negative. Positive expression must be positive at the cell and tissue specific antigenic sites. The human placenta, human liver cancer, human lung cancer, human colon cancer, human breast cancer, human esophagus, human esophageal cancer, and human tonsil tissue were stained with a rabbit monoclonal antibody against human CDKN1A_p21CIP1, and the negative samples were not stained with brain.

Among them, fig. 7 shows the staining behavior in human esophageal cancer tissue. The results show that: the rabbit monoclonal antibody for resisting the human CDKn1A_p21CIP1 is positioned on the cell nucleus, the dyeing positioning is accurate, the dyeing is clear, the nonspecific dyeing is avoided, and the background is clean.

By combining the immunoblotting result, the anti-human CDKn1A_p21CIP1 rabbit monoclonal antibody specifically recognizes human CDKn1A_p21CIP1 protein, has no nonspecific staining, and effectively avoids false positive results.

In addition, it is worth explaining that: the rabbit monoclonal antibody aiming at the human CDKN1A_p21CIP1 protein belongs to a pathological antibody, and compared with MAB-0235 (clone number DCS-60.2, murine monoclonal antibody) which is a commercial Michael company product, the positive anastomosis rate is 100%. The recombinant rabbit monoclonal antibody has the advantages of high affinity, high stability and the like, can meet the WB application, has the specificity verified by KO, and has wider application range and market value.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rabbit monoclonal antibody aiming at human CDKn1A_p21CIP1 protein is characterized in that the sequences of light chain complementarity determining regions of the rabbit monoclonal antibody aiming at human CDKn1A_p21CIP1 protein are respectively shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, and the sequences of heavy chain complementarity determining regions are respectively shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

2. The rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein according to claim 1, wherein the sequence of the light chain variable region of the rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein is shown in SEQ ID No. 2 and the sequence of the heavy chain variable region is shown in SEQ ID No. 7.

3. The rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein according to claim 2, wherein the full length sequence of the light chain of the rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein is shown in SEQ ID No. 1 and the full length sequence of the heavy chain is shown in SEQ ID No. 6.

4. A nucleotide sequence encoding the rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein of any one of claims 1 to 3.

5. An expression vector or host comprising the nucleotide sequence of the rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein of claim 4.

6. Use of a rabbit monoclonal antibody directed against human cdkn1a_p21cip1 protein according to any one of claims 1 to 3 for the preparation of a reagent for detecting human cdkn1a_p21cip1 protein.

7. The use according to claim 6, wherein the method of detection is immunodetection.

8. The use according to claim 7, wherein the immunodetection is at least one of immunohistochemistry, immunoblotting, immunoprecipitation.

9. A method of preparing a rabbit monoclonal antibody directed against human cdkn1a_p21cip1 according to any one of claims 1 to 3, comprising the steps of:

separately loading genes encoding the rabbit monoclonal antibodies against human cdkn1a_p21cip1 of any one of claims 1 to 3 into expression vectors, and transfecting a host;

culturing to obtain a supernatant containing the rabbit monoclonal antibody;

purifying to obtain the final product.