CN117761310A - Deoxycytidine kinase phosphorylating antibody preparation and application thereof in gemcitabine curative effect prediction - Google Patents
Deoxycytidine kinase phosphorylating antibody preparation and application thereof in gemcitabine curative effect prediction Download PDFInfo
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Abstract
The invention relates to the biomedical field, in particular to application of a reagent for detecting the phosphorylation level of a deoxycytidine kinase (DCK) S74 locus in tumor tissues in preparation of a gemcitabine drug effect evaluation reagent and a companion diagnostic reagent kit, and preparation of DCK S74 locus phosphorylated polyclonal and monoclonal antibodies. The application of specific antibodies to detect the phosphorylation level of DCK S74 site in tumor cells and tissues can effectively evaluate the catalytic activity of DCK and the therapeutic effect of gemcitabine. The invention prepares the antibody specifically recognizing DCK S74 locus phosphorylation modification, and has good immunoblotting and immunohistochemical detection energy efficiency. The invention provides the polyclonal and monoclonal antibodies for detecting DCK S74 locus phosphorylation, which can be used for predicting the curative effect of gemcitabine and is easy for basic scientific research and clinical popularization and use.
Description
Technical Field
The invention relates to the biomedical field, in particular to design and preparation of deoxycytidine kinase DCK S74 locus phosphorylating polyclonal and monoclonal antibodies, and evaluation of anti-tumor curative effect of gemcitabine by immunoblotting or immunohistochemical detection by using the antibodies.
Background
Gemcitabine (Gemcitabine) is a deoxycytidine-like chemotherapeutic drug that is widely used for the first-line or super-indication treatment of various solid tumors, including pancreatic ductal adenocarcinoma, biliary tract system tumors, breast cancer, ovarian cancer, non-small cell lung cancer, and the like (Binenbaum et al 2015). Despite the great clinical success of gemcitabine, there are still over 70% of patients with advanced cholangiocarcinoma and over 90% of patients with pancreatic cancer that are poorly responsive to gemcitabine. How to predict the efficacy of gemcitabine-based first-line chemotherapy regimens and to develop subsequent second-line or targeted therapies for patients in time is a difficulty in current advanced cholangiocarcinoma and pancreatic cancer therapies. The development of biomarkers that can predict the efficacy of gemcitabine can avoid delays in disease conditions due to ineffective treatment of the first-line regimen, and can improve the treatment level of advanced tumors.
Gemcitabine exerts an antitumor effect through three steps: the first step is ingestion, and gemcitabine first enters the cell from human nucleoside transporter (human nucleoside transorters, hNTs) on the cell membrane surface; gemcitabine has been shown to be taken up into cells primarily through hENT1 and secondarily through hENT2, hCNT1 and hCNT3 (Ritzel, m.w., ng, a.m., yao, s.y., graham, k., loewen, s.k., smith, k.m., hyde, r.j., karkinski, e., cass, c.e., baldwin, s.a., et al (2001), recent molecular advances in studies of the concentrative Na + -dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT 3 and mCNT 3) broadly selective for purine and pyrimidine nucleosides (system cib), mol Membr Biol 18, 65-72). The second step is activation, and gemcitabine enters cells and is subjected to stepwise phosphorylation to finally form an activated metabolite, nucleoside triphosphates (dFdCTP); wherein the first step is phosphorylated by deoxycytidine kinase (deoxycytidine kinase, DCK) to a critical rate limiting step of the overall metabolic process (Ohhashi, s., ohuchida, k., mizumoto, k., fujita, h., egami, t., yu, j., toma, h., sadatomi, s., nagai, e., and Tanaka, m. (2008) Down-regulation of deoxycytidine kinase enhances acquired resistance to gemcitabine in pancreatic cancer. Anticancer Res 28, 2205-2212). The third step is the incorporation of DNA as a deoxycytidine analog, gemcitabine, by competing with intracellular deoxycytidine (dC), binds to DNA such that DNA synthesis is inhibited and double strand breaks, resulting in cell death (de Sousa Cavalcante, l., and Monteiro, g. (2014). Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Because of the core activation of DCK in gemcitabine drug metabolism, detection of DCK activity can predict the clinical efficacy of gemcitabine.
DCK is the rate-limiting enzyme of the deoxynucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside-like antitumor and antiviral drugs, such as gemcitabine, fludarabine, cladribine, zalcitabine and lamidine (De Clercq, e. (2004) Antiviral drugs in current clinical use.j Clin Virol 30,115-133.Galmarini, c.m., mackey, j.r., and Dumontet, c. (2002) Nucleoside analogues and nucleobases in cancer treatment.Lancet Oncol 3,415-424.Pettitt, a.r. (2003) Mechanism of action of purine analogues in chronic lymphocytic leukaemia.br J haemal 121, 692-702). However, DCK rarely undergoes mutation, deletion or expression disorder in tumors, and merely detecting the protein expression level of DCK to predict the efficacy of gemcitabine may be unsatisfactory. Notably, the catalytic activity of DCK is regulated by autophosphorylation modifications, including sites T3, S11, S15, and S74; wherein S74 is the primary phosphorylation site; the phosphorylation of the site can obviously promote the affinity of DCK and various substrates such as gemcitabine and the like, and promote the catalytic activity of DCK and the drug effect of gemcitabine; in addition, DCK S74 sites may be phosphorylated in response to a variety of drug stimuli including gemcitabine (Smal, c., vertommen, d., bertrand, l., ntamashikiro, s., rider, m.h., van de new, e., and Bontemps, f. (2006) Identification of in vivo phosphorylation sites on human deoxycytidine kinase, rotor of Ser-74in the control of enzyme activity.J Biol Chem 281,4887-4893. Ethong, r., xin, r., chen, z., liang, n., liu, y., ma, s., and Liu, x (2016) in Radiation-Induced Cell de ath, int J Mol Sci 17); therefore, the development of specific antibodies, detection of the phosphorylation level of the DCK S74 site by immunoblotting or immunohistochemistry, can conveniently and effectively evaluate the catalytic activity of DCK, and further predict the therapeutic effect of gemcitabine. Currently, commercial antibodies recognizing DCK S74 site phosphorylation are not available on the market, and there is also a lack of reagents or kits for judging gemcitabine efficacy by detecting DCK S74 site phosphorylation. Furthermore, in tumor clinical cohorts, the correlation between DCK S74 site phosphorylation and gemcitabine chemotherapy effects has not been experimentally verified.
Disclosure of Invention
The invention aims to provide a novel specific recognition DCK S74 locus phosphorylation modified antibody, which has good immunoblotting and immunohistochemical detection energy efficiency; the invention also provides a novel method for predicting the anti-tumor curative effect of the gemcitabine, namely, the specific antibody is used for detecting the DCK S74 locus phosphorylation level in tumor tissues through immunohistochemistry, so that the curative effect of the gemcitabine can be effectively predicted. The novel antibody and the drug effect evaluation method provided by the invention can be used for predicting the development of gemcitabine curative effect reagents or accompanying diagnostic kits, and are beneficial to clinical popularization and use.
According to the invention, two polypeptides containing DCK S74 site phosphorylation modification are synthesized, hemocyanin (KLH) is coupled as an antigen, new Zealand rabbits are mixed and immunized, and a rabbit polyclonal antibody (p-DCK S74 polyclonal antibody) capable of specifically recognizing the phosphorylated peptide and DCK S74 phosphorylated protein is obtained by purification. The present invention demonstrates that the resulting rabbit polyclonal antibodies can effectively detect DCK S74 phosphorylation levels in cells and tissues. In a preferred embodiment of the invention, 98 cholangiocarcinoma patients treated with gemcitabine chemotherapy are classified into two groups, DCK S74 phosphorylation high-expression and low-expression, by immunohistochemical scoring using the specific antibody; combining imaging pathology assessment and prognosis survival statistical analysis, it is demonstrated that patients in the high expression group have better responsiveness to gemcitabine treatment, and longer survival without disease progression and overall survival.
Furthermore, two polypeptides containing S74 site phosphorylation modification are synthesized, hemocyanin (KLH) is coupled as an antigen, mice are mixed and immunized, and a mouse monoclonal antibody (p-DCK S74monoclonal antibody) meeting the requirements is obtained through serum titer and immunoblotting verification, hybridoma cell fusion, positive monoclonal cell line construction and antibody purification, wherein the cloning number is 52-D4-A2.
In conclusion, the invention provides the polyclonal and monoclonal antibodies specifically recognizing DCK S74 locus phosphorylation modification, has good immunoblotting and immunohistochemical detection energy efficiency, can be used for predicting the curative effect of gemcitabine, and is easy for basic scientific research and clinical popularization and use.
In a first aspect of the invention, there is provided the use of a reagent for detecting the phosphorylation level of the DCK S74 site in tumour tissue in the preparation of a gemcitabine potency assessment reagent and accompanying diagnostic kit.
Further, the reagent for detecting the DCK S74 locus phosphorylation level in the tumor tissue is a specific antibody for recognizing DCK S74 locus phosphorylation modification.
Further, the specific antibody is a polyclonal antibody, a monoclonal antibody or a nano antibody which specifically recognizes DCK S74 site phosphorylation modification, and the like.
Further, the application is to detect the phosphorylation level of DCK S74 site in pathological specimens of tumor patients by an immunohistochemical method, and evaluate the staining result, namely the staining score of immunohistochemistry; tumor patients were classified into DCK S74 phosphorylating high-low expression groups according to the score, and patients in DCK S74 phosphorylating high-expression groups had good responsiveness to gemcitabine treatment, while patients in low-expression groups had poor responsiveness to gemcitabine treatment.
In a second aspect of the invention, there is provided a polyclonal antibody specifically recognizing phosphorylation modification of DCK S74 site, said polyclonal antibody being prepared by: by synthesis of peptide fragment 1: cys+eeltms (p) QKNGG and peptide fragment 2: TMS (p) QKNGGNVLQMMY +cys, and conjugated hemocyanin as antigen, and mixing to immunize new zealand rabbits; purification is required to obtain an antibody titer greater than 1:64000, and specifically recognizes phosphorylated peptide segments and DCK S74 phosphorylated proteins.
Through immunoblotting detection, the invention discovers that the prepared p-DCK S74 polyclonal can effectively identify DCK S74 phosphorylated polypeptide (p-Seq 1), but can not detect non-phosphorylated p-DCK S74 polypeptide (Seq 1); after treatment of the phosphorylated DCK S74 polypeptide (p-Seq 1) with lambda phosphatase (lambda PPase), it was not recognized by the p-DCK S74 polyclonal antibody (FIG. 1D); likewise, the p-DCK S74 antibody recognizes wild-type DCK, but not the non-phosphorylated DCK S74A mutant (fig. 1E). The result proves that the p-DCK S74 antibody prepared by the invention has strong specificity and high sensitivity, can only identify the phosphorylation of the wild DCK S74 site, and can be used for subsequent DCK S74 phosphorylation detection.
In a third aspect of the invention, there is provided a murine monoclonal antibody specifically recognizing the phosphorylation modification of DCK S74 site, clone number 52-D4-A2; the preparation method of the monoclonal antibody comprises the following steps: by synthesis of peptide fragment 1: cys+eeltms (p) QKNGG and peptide fragment 2: TMS (p) QKNGGNVLQMMY +cys, and conjugated hemocyanin as antigen, mixing immunized mice, and after serum titer and immunoblotting verification, selecting spleen of positive mice for cell fusion; positive hybridoma cells are obtained through HAT culture medium screening, the positive hybridoma cells are subcloned through a limiting dilution method to obtain monoclonal cell strains, 2-3 rounds of indirect ELISA screening are carried out during each subcloning period to obtain positive monoclonal cell strains meeting requirements, and p-DCK S74monoclonal antibodies are obtained through purification, wherein the cloning number is 52-D4-A2.
The invention proves that the obtained mouse monoclonal antibody can effectively detect the DCK S74 phosphorylation level in cells and tissues; immunoblotting detection shows (figure 4) that the obtained murine monoclonal antibody has higher specificity than the rabbit polyclonal antibody, the target band is clear, and the nonspecific band is less; furthermore, immunohistochemical detection showed (fig. 5) that the obtained murine monoclonal antibodies had less nonspecific staining in the cytoplasm and reduced background interference compared to the aforementioned rabbit polyclonal antibodies; the above experimental data show that the obtained murine monoclonal antibodies are superior to the rabbit polyclonal antibodies described above.
Furthermore, the antibody specifically recognizing the phosphorylation modification of the DCK S74 site can be optimized, modified, combined with other auxiliary agents and used for detecting the DCK S74 phosphorylation level in tumor cells and tissues in various modes. In a preferred embodiment of the invention, detection by immunoblotting and immunohistochemical detection is preferred; the effective detection dose of the antibody of the invention can be a dilution ratio of 1:10-1:100000, more preferably a dilution ratio of 1:100-1:1000.
The method for specifically recognizing DCK S74 locus phosphorylation antibody and predicting the anti-tumor curative effect of the decitabine can be used for the concomitant diagnosis and identification of tumor drugs, and is preferably used for bile duct cancer, pancreatic duct adenocarcinoma, cervical cancer, ovarian cancer, breast cancer and non-small cell lung cancer.
In a fourth aspect of the invention, there is provided a kit for predicting the anti-tumor efficacy of gemcitabine, said kit comprising a specific antibody as described above which recognizes the phosphorylation modification of DCK S74 site. Namely, the specific antibody can be used for detecting the DCK S74 site phosphorylation level in tumor tissues through immunohistochemistry, so that the curative effect of the gemcitabine can be effectively predicted. Is realized by DCK S74 site phosphorylation immunohistochemical staining scoring combined with clinical data analysis; in a preferred embodiment, the present invention demonstrates that patients with cholangiocarcinoma in the DCK S74 phosphorylated high expressing group are more responsive to gemcitabine treatment, with longer survival without disease progression and overall survival.
In a fifth aspect of the present invention, a novel method for predicting the anti-tumor therapeutic effect of gemcitabine is provided, namely, the therapeutic effect of gemcitabine can be effectively predicted by detecting the phosphorylation level of the DCK S74 site in tumor tissues through immunohistochemistry by using the specific antibody.
Further, the key technology of the novel method for predicting the anti-tumor therapeutic effect of the gemcitabine provided by the invention is to detect the DCK S74 locus phosphorylation level in tumor cells and tissues, including but not limited to detection by methods such as immunohistochemistry, immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay, quantitative mass spectrometry and the like.
The invention has the advantages that:
1. gemcitabine is a deoxycytidine-like prodrug that needs to be phosphorylated for activation by DCK, whose catalytic activity is affected by the phosphorylation modification at the S74 site itself. The application of specific antibodies to detect the phosphorylation level of DCK S74 site in tumor cells and tissues can effectively evaluate the catalytic activity of DCK and the therapeutic effect of gemcitabine.
2. The invention provides an antibody for detecting DCK S74 locus phosphorylation, which can be used for predicting the curative effect of gemcitabine and is easy for basic scientific research and clinical popularization and use. According to the invention, 2 DCK phosphorylated polypeptides are designed for immunization, so that polyclonal and monoclonal antibodies which specifically identify DCK S74 locus phosphorylation modification are prepared, and the immune blotting and immunohistochemical detection energy efficiency is good.
3. The invention provides a kit and a method for specifically recognizing DCK S74 site phosphorylation modified antibody and predicting gemcitabine curative effect, which can be used for evaluating gemcitabine curative effect reagent or developing a diagnosis kit, avoid disease delay caused by ineffective first-line chemotherapy treatment based on gemcitabine, enable tumor patients to obtain subsequent second-line treatment in time, improve the treatment level of late tumor patients, and are beneficial to clinical popularization and use. Preservation information of biological material samples:
preservation unit: china Center for Type Culture Collection (CCTCC)
Address: university of Chinese, wuhan and Wuhan
Preservation date: 2023, 10, 11 days
Preservation number: CCTCC NO: C2023306
Classification naming: hybridoma cell line pDCK (Hybridoma cell line pDCK)
Drawings
FIG. 1 shows the preparation and validation of DCK S74 site phosphorylated rabbit polyclonal antibody (p-DCK S74). Wherein A shows DCK phosphorylated peptide fragment 1 (p-Seq 1: cys+EELTMS (p) QKNGG) and peptide fragment 2 (p-Seq 2: TMS (p) QKNGGNVLQMMY +Cys), and the respective corresponding non-phosphorylated peptide fragments (Seq 1 and Seq 2) were synthesized for antigen immunization. B shows a flow chart of mixing 2 phosphorylated polypeptide antigens to immunize 2 New Zealand white rabbits, purifying the antibodies after verifying the potency of antiserum, and screening to obtain qualified p-DCK S74 polyclonal antibodies. C shows the titers of the p-DCK S74 antibody prepared by ELISA detection in combination with DCK phosphorylated peptide fragment p-Seq1 and non-phosphorylated peptide fragment Seq 1. D shows the recognition capacity of the p-DCK S74 antibody prepared by immunoblotting detection on Seq1, p-Seq1 and p-Seq1 after treatment with lambda PPase. E shows the recognition ability of the prepared p-DCK S74 antibody to DCK wild type (DCK WT) and S74 site mutated DCK (DCK S74A) transfected in HEK-293T cells by immunoblotting.
FIG. 2 shows immunoblotting and immunohistochemical detection energy efficiency of the prepared p-DCK S74 rabbit polyclonal antibody. Wherein A shows DCK phosphorylation levels in HCCC-9810, RBE, SK-CHA-1 and CCLP1 cholangiocarcinoma cells after gemcitabine (0,5,20. Mu.M, 24 h) treatment using the prepared p-DCK S74 rabbit polyclonal antibody by immunoblotting. B shows the detection of DCK phosphorylation levels in gemcitabine treated and control cholangiocarcinoma PDX transplanted tumor tissues (CC 1, CC2, CC13, CC17, CC 21) by immunohistochemical method using the prepared p-DCK S74 rabbit polyclonal antibody; gemcitabine is injected intraperitoneally, 10mg/kg, 3 times per week for 5 weeks. Scale bar: 50 μm.
FIG. 3 shows the detection of DCK phosphorylation levels in biliary tract cancer cohorts using prepared p-DCK S74 rabbit polyclonal antibodies, demonstrating that patients with high DCK phosphorylation levels are more responsive to gemcitabine treatment. Waterfall plot a shows tumor burden change from onset to optimal objective response in 98 patients with advanced cholangiocarcinoma following gemcitabine-based first-line chemotherapy; representative pictures of p-DCK S74 staining are shown below, scale bar: 50 μm; scoring according to the degree of staining of p-DCK S74, and dividing the patients into a p-DCK S74 low expression group (less than or equal to 1 point) and a p-DCK S74 high expression group (greater than 1 point); the dashed line above the X-axis represents a 20% increase in the sum of the diameters of the target lesions from the minimum, and the dashed line below the X-axis represents a 30% decrease in the sum of the diameters of the target lesions from the baseline; PD: disease progression, SD: stable disease and partial alleviation of PR. And B, counting the curative effect evaluation results and the proportion of patients with bile duct cancer with high or low expression of P-DCK S74 by a table and a histogram, wherein the P value is obtained by chi-square test. Disease Progression Free Survival (PFS) and total survival (OS) of cholangiocarcinoma patients were analyzed by Kaplan-Meier survival in C, D; cholangiocarcinoma patients receiving gemcitabine chemotherapy were grouped according to p-DCK S74 staining scores; p values were obtained from the log-rank test.
FIG. 4 shows the detection effect of the prepared DCK S74 site phosphorylated murine monoclonal antibody (p-DCK S74monoclonal antibody) in immunoblots. A shows the recognition ability of p-DCK S74 murine monoclonal antibody (52-D4-A2) prepared by immunoblotting detection on DCK wild type (DCK WT) and S74 site mutated DCK (DCK S74A) transfected into HEK-293T cells. B shows that after RBE cholangiocarcinoma cells were treated with gemcitabine (5. Mu.M, 6 h), DCK phosphorylation levels in the cells were detected using the prepared p-DCK S74 rabbit polyclonal antibody and murine monoclonal antibody, respectively.
FIG. 5 shows immunohistochemical staining of the prepared DCK S74 murine monoclonal antibody and rabbit polyclonal antibody in bile duct cancer tumor tissue.
Detailed Description
The following provides a detailed description of embodiments of the present invention with reference to examples. In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
DCK S74 site phosphorylation modified peptide segment 1 (p-Seq 1: cys+EELTMS (p) QKNGG) and peptide segment 2 (p-Seq 2: TMS (p) QKNGGNVLQMMY +Cys), respectively corresponding to the non-phosphorylated peptide segments Seq1 (CEELTMSQKNGG, SEQ ID NO: 1) and Seq2 (TMSQKNGGNVLQMMYC, SEQ ID NO: 2) by Wohazepin biosynthesis; wild type DCK plasmid (DCK-WT) was purchased from Beijing Yiqiao Shenzhou Co, and DCK S74A mutant plasmid was constructed from wild type plasmid mutation and verified by sequencing; beta-actin (AC 026) and GAPDH (AC 002) antibodies were purchased from ABclonal, and DCK antibodies were purchased from Proteintech Group (17758-1-Ig); the p-DCK S74 rabbit polyclonal antibody and the mouse monoclonal antibody (52-D4-A2) are self-made by the inventor and are subjected to specific detection; lambda phosphatase (lambda PPase) was purchased from NEB company; gemcitabine (GEM) is available from Selleck corporation; protease inhibitors and phosphatase inhibitor cocktails were purchased from Topscience company; human bile duct cancer cells RBE, HCCC-9810, CCLP1, and human embryonic kidney epithelial cells HEK-293T were purchased from Shanghai cell Bank of China academy of sciences; female nude mice (SPF grade) of BALB/c (nu/nu) 6 weeks old were purchased from Shanghai BiKai organism; human cholangiocarcinoma cell SK-CHA-1 cells; human cholangiocarcinoma tumor xenograft model (Patient-derived tumor xenograft, PDX) (cell line culture and human cholangiocarcinoma PDX model building methods see literature Jiang, t.y., pan, y.f., wan, z.h., lin, y.k., zhu, b., yuan, z.g., ma, y.h., shi, y.y., zeng, t.m., dong, l.w., et al (2020) PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma. Other reagents or equipment used were conventional products commercially available without the manufacturer's knowledge.
All bile duct cancer tissue samples used in the invention are derived from a third affiliated hospital of naval medical university; samples and clinical information were approved using review by the ethics committee of the hospital and informed consent was obtained in writing from the patient. The specific embodiment of the invention comprises a clinical bile duct cancer queue subjected to gemcitabine chemotherapy, and the group-entering standard is as follows: (1) pathologically or cytologically diagnosed as cholangiocarcinoma; (2) RECIST standard measurable assessment; (3) between 18 and 75 years of age; (4) WHO/ECOG physical strength score 0-2 points; (5) Distant metastasis has occurred when the lesion is unresectable or diagnosed and has not received any systemic treatment; (6) informed consent; (7) Treated by gemcitabine, the dosage of the medicine is 1000mg/m 2 Intravenous drip (days 1, 8). The primary endpoint is Progression-free survival (PFS); the secondary endpoint was total survival (Overall survivin,OS), objective response rate (Overall response rate, ORR), disease control rate (Disease control rate, DCR), safety, and the like. Using a chi-square test to evaluate the correlation of DCK phosphorylated protein expression with gemcitabine potency, drawing a Kaplan-Meier survival curve with GraphPad Prism (version 7.0), and comparing the differences in survival curves with Log-rank method; p is p<A difference of 0.05 is considered statistically significant.
Example 1: preparation and verification of rabbit polyclonal antibody (p-DCK S74 antibody) specifically recognizing DCK S74 site phosphorylation modification
The catalytic activity of DCK is mainly controlled by the phosphorylation of S74 site; however, there is a lack of ready-made DCK S74 phosphorylated antibodies on the market. The present invention predicts possible epitopes based on DCK protein sequences, and designs two pairs of DCK S74 phosphorylated and non-phosphorylated peptides (Seq 1 and p-Seq1, seq2 and p-Seq 2) for subsequent immunization (fig. 1A). Each of p-Seq1 and p-Seq2 was synthesized to 20mg, with a purity of 95% or more, 10mg of conjugated hemocyanin (KLH) as antigen, and the remaining 10mg of bare peptide was used for detection and purification. According to the invention, 2 phosphorylated polypeptide antigens are mixed to immunize 2 New Zealand white rabbits; detecting the serum titer after the third immunization and the fourth immunization by an indirect ELISA method; when the potency is greater than 1: at 32000, antisera were collected by blood collection and antigen-specific antibody purification was performed, requiring a purified antibody titer of greater than 1:64000 (FIG. 1B). ELISA detection demonstrated that the antibody had high binding titers to DCK S74 phosphorylated peptide (p-Seq 1) and low binding titers to non-phosphorylated peptide (Seq 1), suggesting that the antibody was highly specific (FIG. 1C). Subsequently, the invention discovers that the prepared p-DCK S74 polyclonal antibody can effectively recognize DCK S74 phosphorylated polypeptide (p-Seq 1) through immunoblotting detection, and can not detect non-phosphorylated p-DCK S74 polypeptide (Seq 1); after treatment of the phosphorylated DCK S74 polypeptide (p-Seq 1) with lambda phosphatase (lambda PPase), it was not recognized by the p-DCK S74 polyclonal antibody (FIG. 1D); likewise, the p-DCK S74 antibody recognizes wild-type DCK, but not the non-phosphorylated DCK S74A mutant (fig. 1E). The results prove that the prepared p-DCK S74 antibody has strong specificity and high sensitivity, can only recognize the phosphorylation of the wild DCK S74 site, and can be used for subsequent DCK S74 phosphorylation detection.
Example 2: the prepared p-DCK S74 rabbit polyclonal antibody can be used for detecting DCK S74 site phosphorylation level in tumor cells and tissues
DCK S74 site phosphorylation is activated by feedback of its substrates, such as deoxycytidine nucleotides and gemcitabine. Based on the detection energy efficiency of the p-DCK S74 rabbit polyclonal antibody on phosphorylated DCK is verified in tumor cells and tissues by immunoblotting and immunohistochemical techniques.
Immunoblotting detection is mainly performed according to the following steps: (1) preparing a protein sample: collecting 20 μM gemcitabine-treated and control cholangiocarcinoma cells, including cell lines in HCCC-9810, RBE, SK-CHA-1, and CCLP 14; lysing with RIPA buffer (bi yun) and adding protease inhibitor and phosphatase inhibitor mixture (Topscience) in advance; carrying out ultrasonic, centrifugal and BCA quantitative analysis on a protein sample, and preparing the protein sample into an equal concentration protein sample in a 1 XSDS loading buffer solution; (2) Protein samples were subjected to SDS-PAGE electrophoresis, membrane transfer and membrane blocking, then incubated with a specific primary antibody (4 ℃ C., overnight), then incubated with a fluorescein-conjugated secondary antibody (37 ℃ C., 60 minutes), and after washing, fluorescence signals were detected using an Aldrich fluorescence scanner (Li-Cor); (3) The dilution ratio of the p-DCK S74 rabbit polyclonal antibody of the immunoblotting is 1:1000, and the dilution ratio of the secondary antibody is 1:5000. The experimental results show that: the p-DCK S74 rabbit polyclonal antibody can detect gemcitabine-induced phosphorylated DCK protein in a variety of cholangiocarcinoma cell lines, and this recognition is specific (fig. 2A).
The immunohistochemical detection is mainly carried out according to the following steps: (1) Fixing the human cholangiocarcinoma PDX tissue treated by gemcitabine and controlled by gemcitabine by 4% neutral formaldehyde for 24 hours, and then dehydrating and embedding paraffin; paraffin samples were cut into 4 μm thick sections and adsorbed onto polylysine coated slides; (2) Before the staining experiment, the tissue slice is put into a 60 ℃ oven to be baked for 1 hour, then conventional immunohistochemical staining is carried out, the steps comprise dewaxing, peroxidase inactivation, antigen retrieval, sealing, primary antibody incubation, secondary antibody incubation, DAB color development, hematoxylin staining and tap water reverse blue, and finally the slice is dehydrated; (3) The dilution ratio of the p-DCK S74 rabbit polyclonal antibody in immunohistochemistry is 1:200. Experimental results show that the staining of the p-DCK S74 rabbit polyclonal antibody is mainly concentrated in the nucleus and is consistent with the subcellular localization of DCK (FIG. 2B); in addition, the staining of the p-DCK S74 rabbit polyclonal antibody was significantly increased in gemcitabine-treated PDX tumor tissue, suggesting that gemcitabine may induce an increased level of DCK phosphorylation, further confirming the specificity and efficacy of the p-DCK S74 rabbit polyclonal antibody at the tissue level.
Example 3: high p-DCK S74 expression in cholangiocarcinoma patients suggests better gemcitabine drug responsiveness
The experiment proves that the prepared p-DCK S74 rabbit polyclonal antibody can effectively identify phosphorylated DCK, so that the catalytic activity of the DCK is reflected, and the clinical value of gemcitabine drug sensitivity is potentially evaluated. Subsequently, we validated the above assumption using this antibody in 98 patients with advanced cholangiocarcinoma who received a first-line gemcitabine-based chemotherapy regimen. According to the invention, the expression of p-DCK S74 in a cholangiocarcinoma sample is evaluated through immunohistochemical staining, and is evaluated by two independent pathologists, and scoring is carried out according to the dyeing degree of the p-DCK S74, wherein the scoring standard is as follows: randomly selecting five fields of view on each slice of all specimens while considering staining intensity and frequency of positive cells; the dyeing intensity is divided into: 0 is divided into no coloring, 1 is divided into pale yellow particles, 2 is divided into yellow brown particles, and 3 is divided into dark brown particles; frequencies of positive cells are divided into: 0 (less than 5%), 0.25 (5% -25%), 0.5 (6% -50%), 0.75 (51% -75%) and 1 (greater than 75%). When the staining was uneven, each area was scored independently and the results were added. For example, a specimen containing 75% of 2-part stained tumor cells (2×0.75=1.5) and 25% of 1-part stained tumor cells (1×0.25=0.25) has a final score of 1.5+0.25=1.75; in the statistical analysis, the p-DCK S74 staining score is less than or equal to 1 and is low in expression, and the score is more than 1 and is high in expression.
Representative pictures of p-DCK S74 staining scores 0,1, 2 and 3 are shown in fig. 3A. In the bile duct cancer chemotherapy queue, total p-DCK S74 is expressed in 60 cases, and total p-DCK S74 is expressed in 38 cases. Waterfall plots of tumor burden change showed that patients with p-DCK S74 high expression group (p-DCK S74 high) were more likely to acquire disease Stabilization (SD) and Partial Remission (PR) after gemcitabine chemotherapy (fig. 3A). The objective response rate of the p-DCK 74 high expression group was 42.1% (16/38), the disease control rate was 86.8% (33/38), and was far higher than 15.0% (9/60) and 65.0% (39/60) of the p-DCK 74 low expression group (FIG. 3B). Furthermore, PFS (p=0.006) and OS (p=0.034) were significantly longer in p-DCK S74 high expression group patients than in p-DCK S74 low expression group; the above results demonstrate that patients in the p-DCK S74 high expression group are more responsive to gemcitabine treatment. In conclusion, the p-DCK S74 rabbit polyclonal antibody prepared by the invention can be used for predicting the clinical curative effect of gemcitabine and developing a diagnosis kit.
Example 4: the prepared p-DCK S74 murine monoclonal antibody (52-D4-A2) is superior to the rabbit polyclonal antibody obtained in Western immunoblotting and immunohistochemical detection.
The invention is characterized by synthesizing a peptide segment 1: cys+eeltms (p) QKNGG and peptide fragment 2: TMS (p) QKNGGNVLQMMY +cys, and conjugated hemocyanin as antigen, mixing immunized mice, and after serum titer and immunoblotting verification, selecting spleen of positive mice for cell fusion; positive hybridoma cells are obtained through HAT culture medium screening, the positive hybridoma cells are subcloned through a limiting dilution method to obtain monoclonal cell strains, 2-3 rounds of indirect ELISA screening are carried out during each subcloning period to obtain positive monoclonal cell strains meeting requirements, and p-DCK S74monoclonal antibodies are obtained through purification, wherein the cloning number is 52-D4-A2.
Subsequently, the present invention found that the prepared p-DCK S74 murine monoclonal antibody can effectively recognize wild-type DCK, but not non-phosphorylated DCK S74A mutant, by immunoblotting detection (FIG. 4A); in addition, compared with the obtained p-DCK S74 rabbit polyclonal antibody, the p-DCK 74 mouse monoclonal antibody has clear target bands and fewer nonspecific bands when detecting DCK phosphorylation levels in RBE cells stimulated by gemcitabine (figure 4B), which suggests that the p-DCK S74 mouse monoclonal antibody has better immunoblotting detection energy efficiency.
Furthermore, the invention respectively applies the prepared p-DCK S74 murine monoclonal antibody and rabbit polyclonal antibody to immunohistochemical staining of bile duct cancer tumor tissues; the result shows that the obtained p-DCK S74 mouse monoclonal antibody staining is mainly concentrated in the cell nucleus and is consistent with the subcellular localization of DCK reported in the literature; less nonspecific staining of the p-DCK S74 murine monoclonal antibody in the cytoplasm compared to the rabbit polyclonal antibodies described previously, reduced background interference (fig. 5); the experimental data prove that the obtained mouse monoclonal antibody has better immunohistochemical detection energy efficiency and is superior to the rabbit polyclonal antibody obtained in the prior art.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (10)
1. The application of a reagent for detecting DCK S74 locus phosphorylation level in tumor cells and tissues in preparing a gemcitabine drug efficacy evaluation reagent and a concomitant diagnostic kit.
2. The use according to claim 1, wherein the reagent for detecting DCK S74 site phosphorylation level in tumor cells and tissues is a specific antibody recognizing DCK S74 site phosphorylation modification.
3. The use according to claim 2, wherein the specific antibody is a polyclonal antibody, monoclonal antibody or nanobody specifically recognizing the phosphorylation modification of DCK S74 site.
4. The use according to claim 1, wherein the level of phosphorylation of DCK S74 site in pathological specimens of tumor patients is detected by immunohistochemical methods and the staining results, i.e. the staining score of immunohistochemistry, is assessed; tumor patients were classified into DCK S74 phosphorylating high-low expression groups according to the score, and patients in DCK S74 phosphorylating high-expression groups had good responsiveness to gemcitabine treatment, while patients in low-expression groups had poor responsiveness to gemcitabine treatment.
5. The use according to claim 1, wherein the neoplasm is cholangiocarcinoma, ductal adenocarcinoma of the pancreas, cervical cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.
6. The polyclonal antibody specifically recognizing DCK S74 site phosphorylation modification is characterized in that the preparation method of the polyclonal antibody comprises the following steps: by synthesis of peptide fragment 1: cys+eeltms (p) QKNGG and peptide fragment 2: TMS (p) QKNGGNVLQMMY +cys, and conjugated hemocyanin as antigen, and mixing to immunize new zealand rabbits; purification is required to obtain an antibody titer greater than 1:64000, and specifically recognizes phosphorylated peptide segments and DCK S74 phosphorylated proteins.
7. The monoclonal antibody specifically recognizing DCK S74 locus phosphorylation modification is characterized in that the preparation method of the monoclonal antibody comprises the following steps: by synthesis of peptide fragment 1: cys+eeltms (p) QKNGG and peptide fragment 2: TMS (p) QKNGGNVLQMMY +cys, and conjugated hemocyanin as antigen, mixing immunized mice, and after serum titer and immunoblotting verification, selecting spleen of positive mice for cell fusion; positive hybridoma cells are obtained through HAT culture medium screening, the positive hybridoma cells are subcloned through a limiting dilution method to obtain monoclonal cell strains, 2-3 rounds of indirect ELISA screening are carried out during each subcloning period to obtain positive monoclonal cell strains meeting requirements, and p-DCK S74monoclonal antibodies are obtained through purification, wherein the cloning number is 52-D4-A2.
8. The monoclonal antibody of claim 7, wherein the monoclonal antibody is secreted by a hybridoma cell line having a accession number cctcno: C2023306.
9. A kit for predicting the anti-tumor efficacy of gemcitabine, comprising a specific antibody that recognizes the phosphorylation modification of DCK S74 site.
10. The kit of claim 9, wherein the kit predicts the efficacy of gemcitabine by immunohistochemical detection of DCK S74 site phosphorylation in tumor tissue; tumor patients in the DCK S74 site phosphorylated high expression group were more responsive to gemcitabine treatment, and had longer disease progression-free survival and overall survival.
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