Anti-Cx43 Ser368 site phosphorylated specific monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-Cx43 Ser368 site phosphorylated specific monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The specific monoclonal antibody is prepared from a Cx43 Ser368 site phosphorylated antigen peptide immune animal, can be specifically combined with a Cx43 Ser368 phosphorylated form in PASMCs in a pulmonary arterial hypertension specimen, does not have a cross reaction with non-phosphorylated Cx43 or other site phosphorylated forms, has an equilibrium dissociation constant KD of less than or equal to 100 nM, can detect a phosphorylated signal induced by nicotine exposure in a dose-dependent manner, and can be used for preparing the specific monoclonal antibody. And PKC / Cx43-pS368 / MEK / ERK signal axis is used for associating the proliferation, migration, dedifferentiation and cardiopulmonary remodeling degree of the PASMC. The method has the beneficial effects of high specificity, high cell purity, excellent model pertinence, wide mechanism coverage, multiple application scenes, complete verification system and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and contains a specific monoclonal antibody against phosphorylation at the Ser368 site of Cx43, its preparation method and application. Background Technology

[0002] Connexin 43 (Cx43, gene name GJA1) is a core protein constituting gap junction channels and is widely expressed in pulmonary artery smooth muscle cells (PASMCs). Its phosphorylation modification affects PASMC proliferation, phenotypic transformation, migration, and inflammatory response by regulating the ERK1 / 2 signaling pathway, and is a key molecular mechanism for pulmonary hypertension vascular remodeling and cardiopulmonary remodeling.

[0003] Chronic nicotine exposure can dose-dependently induce pulmonary hypertension. With increasing nicotine dose (0.02-2.0 mg / kg·d), mouse weight gain slows, survival time is shortened, and abnormal proliferation of PASMCs and cardiopulmonary remodeling are significantly promoted, upregulating right ventricular systolic pressure and free wall thickness. Current technologies lack phosphorylation site specificity for Cx43 antibodies and cannot quantify the correlation between nicotine exposure dose and signal intensity. Commonly used methods for isolating and culturing PASMCs suffer from low cell purity and long culture periods. Traditional validation models have poor specificity and struggle to exclude interference from other cell types. More importantly, current technologies have not yet clarified the key functional sites and upstream regulatory mechanisms of Cx43 phosphorylation, making it impossible to directly link Cx43 phosphorylation with the core pathological processes of PASMC dedifferentiation and pulmonary artery remodeling. This prevents a closed loop from forming in the "etiology-mechanism-diagnosis" of nicotine-induced pulmonary hypertension.

[0004] Magnetic separation can rapidly separate high-purity PASMCs. Combined with a Tagln-Cre-mediated VSMC-specific Cx43 knockdown model, the function of the Ser368 site can be accurately verified. Bioinformatics prediction tools can clarify the specific binding relationship between PKC and the Ser368 site. This invention combines the above-mentioned technological advantages with the dose-dependent pathological characteristics induced by nicotine to develop highly specific antibodies, overcome the shortcomings of existing technologies, and meet multiple needs in clinical diagnosis, basic research, and drug screening. Summary of the Invention

[0005] The purpose of this invention is to overcome the technical shortcomings of existing Cx43 antibodies, such as insufficient specificity, low purity of PASMCs, poor targeting of traditional validation models, inability to quantify nicotine dose-related signals, and lack of mechanistic correlation. This invention provides a specific monoclonal antibody that can accurately identify PKC-mediated phosphorylation of Cx43 Ser368. It also provides a preparation method and application based on bioinformatics prediction, magnetic separation, and VSMC-specific Cx43 KD mouse validation. Furthermore, it links the ERK1 / 2 signaling pathway to elucidate the mechanisms of PASMC proliferation, inflammatory response, dedifferentiation, and cardiopulmonary remodeling, providing a reliable tool for pulmonary hypertension diagnosis, dose-related risk stratification, PASMC dedifferentiation detection, and drug screening. This invention is applicable to basic research, clinical diagnosis, dose-related risk stratification, PASMC dedifferentiation detection, and drug screening related to pulmonary hypertension (including nicotine-induced pulmonary hypertension), and can link the ERK1 / 2 signaling pathway to elucidate the mechanisms of PASMC proliferation, migration, inflammatory response, and cardiopulmonary remodeling.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a specific monoclonal antibody against phosphorylation at the Cx43 Ser368 site, which is prepared by immunizing animals with a phosphorylated antigenic peptide at the Cx43 Ser368 site. It can specifically bind to the phosphorylated form of Cx43 Ser368 in PASMCs in pulmonary hypertension specimens, does not cross-react with non-phosphorylated Cx43 or other phosphorylated forms, has an equilibrium dissociation constant KD≤100nM, can detect nicotine exposure-induced phosphorylation signals in a dose-dependent manner, and correlates the degree of PASMC proliferation, migration, dedifferentiation, and cardiopulmonary remodeling through the PKC / Cx43-pS368 / MEK / ERK signal axis.

[0007] Furthermore, the Cx43 Ser368 phosphorylated antigenic peptide is based on the Ser368 phosphorylation site of the human Cx43 protein, containing 6 amino acid residues on the N-terminal side, 7 amino acid residues on the C-terminal side, and Ser368 phosphorylation modification. The active amino acid sequence is SEQ ID NO:1. The Ser368 site is a PKC kinase-specific phosphorylation site. According to the NetPhos3.1 and GPS6.0 bioinformatics models, the phosphorylation probability is ≥99.5%, which is significantly higher than other potential phosphorylation sites of the Cx43 protein. The corresponding non-phosphorylated antigenic peptide sequence is SEQ ID NO:2 (C-PSSRASSRASSR-COOH), which is used for specific screening. The purity of the Cx43 Ser368 phosphorylated antigenic peptide is ≥95%, and it is used for immunization after being conjugated with the carrier protein KLH.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned specific monoclonal antibody against phosphorylation at the Cx43 Ser368 site, comprising: Bioinformatics prediction of S0 and Cx43 phosphorylation sites: The NetPhos3.1 model was used to screen potential phosphorylation sites of Cx43 protein, and the GPS6.0 model was used to predict PKC kinase-specific binding sites. The results showed that the phosphorylation probability of the Ser368 site was 99.59%, which is a highly specific target of the PKC family, significantly higher than other potential sites, and Ser368 was identified as the core functional site.

[0009] S1. Verification of Cx43 Ser368 as a functional phosphorylation site: Using Tagln-Cre-mediated VSMC-specific Cx43 gene knockdown mice, Cx43 KD mouse PASMCs were isolated using magnetic separation. Cell purity was verified by α-SMA immunofluorescence, and the knockdown efficiency of Cx43 protein and mRNA was verified by Western blot and qPCR to be ≥70%. Cx43 wild-type and Ser368 site mutant eukaryotic expression plasmids were introduced into cells via liposome-mediated transient transfection. Samples were collected 48-72 h after transfection, and the phosphorylation function of Cx43 Ser368 site and its association with the ERK1 / 2 pathway were verified by immunoprecipitation.

[0010] S2. Design and synthesize antigenic peptides: synthesize phosphorylated antigenic peptides at Cx43 Ser368 site and corresponding non-phosphorylated antigenic peptides, and conjugate them with KLH and BSA respectively to obtain immunogens and screening antigens.

[0011] S3. Animal immunization: C57BL / 6J mice were used as the immunization subjects. They were initially immunized with Freund's complete adjuvant and then given three booster immunizations with Freund's incomplete adjuvant. The serum antibody titer was ≥1:50000 7 days after the last immunization.

[0012] S4. Cell fusion and screening: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a ratio of 1:5 using PEG1500, and screened in HAT / HT medium. Antibody-specific screening was performed using four identification peptides.

[0013] S5. Cloning and Purification: Positive hybridoma cells were cloned using a three-round limiting dilution method. Antibodies were collected by in vitro culture in serum-free medium or by intraperitoneal injection in mice. Protein A affinity chromatography was used for purification to obtain the target antibody.

[0014] Furthermore, in step S1, Cx43 KD mice were used with a C57BL / 6J background, and the targeting sgRNA sequence was SEQ ID NO:3, targeting a key exon in the coding region of the Cx43 gene. PASMCs were isolated from the pulmonary artery media tissue of Cx43 KD mice and cultured in primary culture to the 3rd generation for experiments. The cells in this generation showed a typical long spindle-shaped "peak-valley" morphology, an α-SMA positivity rate of ≥95%, and low basal OPN expression, confirming that they were in a normal contractile phenotype.

[0015] In step S1, the specific operation of separating PASMCs by magnetic separation is as follows: After anesthetizing Cx43 KD mice, blood was removed by injecting 4°C PBS and iron powder suspension at 37°C into the left ventricle in sequence, and pulmonary artery tissue was separated under a dissecting microscope; The pulmonary artery was cut into 1.0-1.5 mm tissue blocks and digested with collagenase IV for 45 minutes. The collagenase was removed by adsorption using a magnetic separator. The cells were washed 2-3 times with M199 medium containing 20% ​​FBS, inoculated, and cultured. The medium was completely changed every 3 days. The cells were passaged when they reached 85%-90% confluence in about 7 days.

[0016] In step S1, the specific transfection operations are as follows: A recombinant lentiviral vector containing the Cx43-WT or Cx43-S368A gene was constructed, co-transfected with the packaging plasmid into 293T cells, and the viral fluid was collected and concentrated to a viral titer ≥1×10⁻⁶. 8 TU / mL; Cx43 KD mouse PASMCs were inoculated into 6-well plates. When the confluence reached 60%-70%, recombinant lentivirus solution was added. The multiple of transfection (MOI) was 10-20. At the same time, 8 μg / mL polybrene was added to enhance the transfection efficiency. After culturing at 37℃ and 5% CO2 for 24 hours, the medium was replaced with fresh medium, and the culture was continued for another 48 hours. Western blot was used to verify Cx43 protein expression, confirming successful reinjection.

[0017] In step S1, the specific verification procedure for verifying the phosphorylation function of the Cx43 Ser368 site and its association with the ERK1 / 2 pathway via immunoprecipitation is as follows: Wild-type mouse PASMCs and Cx43 KD mouse PASMCs without vector were used as controls; After culture, cells were collected, total protein and total RNA were extracted, and Cx43 mRNA expression was detected by qPCR. Western blot was used to verify that the Cx43 protein knockdown efficiency was ≥70% and the expression level was up to standard. At the same time, ERK1 / 2 phosphorylation level and inflammatory factor expression were detected. Immunoprecipitation enriched Cx43 protein, and Western blot analysis with panphosphorylated serine antibody showed that the WT vector-filled group exhibited obvious phosphorylation bands, increased ERK1 / 2 phosphorylation, and upregulated expression of inflammatory factors, while the S368A vector-filled group showed no bands and no significant changes. The endogenous Cx43 phosphorylation signal in PASMCs of Cx43 KD mice without vector introduction was reduced compared with wild type, confirming that Ser368 is a functional phosphorylation site.

[0018] Furthermore, in step S4, the four identified peptides include: Peptides phosphorylated only at the Cx43 Ser368 site; Peptides without any phosphorylation sites; A polypeptide phosphorylated at both Ser368 and Ser365 sites of Cx43; A peptide phosphorylated only at the Cx43 Ser365 site; The screening criteria were: the antibody reacted with the first two antibodies at an OD450nm ≥ 1.0, and reacted with the latter two antibodies at an OD450nm < 0.5.

[0019] A third aspect of the present invention provides the use of the above-described specific monoclonal antibody against Cx43 Ser368 phosphorylation in the preparation of a Cx43 Ser368 phosphorylation detection reagent.

[0020] A fourth aspect of the present invention provides the use of the above-described specific monoclonal antibody against Cx43 Ser368 phosphorylation in the preparation of a formulation for detecting the dedifferentiation of PASMCs; wherein the detection is achieved by associating the expression of the dedifferentiation marker OPN and the shrinkage phenotype marker α-SMA.

[0021] A fifth aspect of the present invention provides the use of the above-mentioned specific monoclonal antibody against Cx43 Ser368 phosphorylation in the preparation of drug formulations for screening the PKC / Cx43-pS368 signaling pathway; wherein the drug alleviates PASMC dedifferentiation and pulmonary artery remodeling by inhibiting Cx43 Ser368 phosphorylation.

[0022] A sixth aspect of the present invention provides a diagnostic / prognostic kit for pulmonary hypertension, characterized in that it comprises the above-mentioned specific monoclonal antibody against Cx43 Ser368 phosphorylation, blocking solution, washing buffer, chromogenic solution, and Cx43 Ser368 phosphorylated peptide standard.

[0023] In summary, the specific monoclonal antibody against Cx43 Ser368 phosphorylation described in this invention is prepared by immunizing animals with a phosphorylated antigenic peptide (SEQ ID NO: 1, sequence: C-PSSRASpSRASSR-COOH) targeting the Cx43 Ser368 site. It can specifically recognize the Cx43 Ser368 phosphorylated form of PASMCs in pulmonary arterial hypertension (especially nicotine-induced) specimens, does not cross-react with non-phosphorylated Cx43 or other phosphorylation sites, and can dose-dependently detect phosphorylation signals at different nicotine exposure levels. The preparation method includes verifying the Ser368 site using Tagln-Cre-mediated VSMC-specific Cx43 KD mice (constructed using CRISPR / Cas9 technology), efficiently isolating and culturing PASMCs (purity ≥98%) using magnetic separation, antigenic peptide synthesis and conjugation, animal immunization, hybridoma cell fusion and screening, antibody purification and identification, and gene complementation through lentiviral transfection. This antibody can accurately detect the phosphorylation level of Cx43 Ser368 in PASMCs through experiments such as Western blot and immunofluorescence, revealing the mechanism by which it regulates the proliferation, migration and inflammatory response of PASMCs, and is associated with the assessment of right ventricular hypertrophy and cardiopulmonary remodeling. It provides a key tool for the early diagnosis, dose-related risk stratification, pathological classification and prognostic assessment of pulmonary hypertension. It solves the technical defects of existing antibodies such as insufficient specificity, low purity of PASMCs isolation and inability to quantify nicotine exposure dose-related signals. The verification results have better accuracy and reproducibility, and have important clinical translational value.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) High specificity: It only recognizes the phosphorylated form of Cx43 Ser368 mediated by PKC and does not cross-react with other sites. Bioinformatics prediction and experimental verification show that its site specificity is significantly better than that of existing antibodies.

[0025] 2) High cell purity: PASMCs are separated by magnetic separation method and verified by OPN / α-SMA phenotype. There is less interference from other cells, and the verification results are more reliable.

[0026] 3) Excellent model targeting: Tagln-Cre mediates VSMC-specific Cx43 KD mice, accurately excluding interference from other cells, which fits the research scenario of pulmonary hypertension.

[0027] 4) Dose-relatedness: It can quantify phosphorylation signals under different nicotine exposure doses, providing a quantitative basis for risk stratification of smoking-related pulmonary hypertension.

[0028] 5) Broad mechanism coverage: It is associated with the PKC / Cx43-pS368 / MEK / ERK signaling axis, inflammatory response, PASMC dedifferentiation and cardiopulmonary remodeling, providing tools for multidimensional research.

[0029] 6) Diverse application scenarios: It meets four major needs: clinical diagnosis, basic research, PASMC dedifferentiation detection and drug screening, and has high clinical translational value.

[0030] 7) Complete verification system: From bioinformatics prediction → gene knockdown verification → inhibitor verification → in vivo and phenotypic correlation, a complete chain of evidence is formed, making the antibody more reliable. Attached Figure Description

[0031] Figure 1 A procedure for identifying VSMC-specific Cx43 KD mice and magnetic separation of PASMCs.

[0032] Figure 2 Immunofluorescence identification of α-SMA in PASMCs.

[0033] Figure 3 Colocalization map of Cx43-pS368 and dedifferentiation markers under nicotine intervention.

[0034] Figure 4 Correlation analysis for the right ventricular hypertrophy index.

[0035] Figure 5 Bioinformatics prediction results for Cx43 phosphorylation sites (NetPhos3.1 GPS6.0).

[0036] Figure 6 The effect of PKC inhibitors on reversing Cx43-pS368 expression and PASMC dedifferentiation, proliferation, and migration. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This invention provides a specific monoclonal antibody against phosphorylation at the Cx43 Ser368 site. It is prepared by immunizing animals with a phosphorylated antigenic peptide at the Cx43 Ser368 site. It can specifically bind to the phosphorylated form of Cx43 Ser368 in PASMCs in pulmonary arterial hypertension (especially nicotine-induced) specimens, without cross-reacting with non-phosphorylated Cx43 or other phosphorylated forms. The equilibrium dissociation constant KD≤100nM. It can dose-dependently detect phosphorylation signals induced by nicotine exposure (0.02-2.0 mg / kg·d). It correlates the proliferation, migration, dedifferentiation, and cardiopulmonary remodeling of PASMCs through the PKC / Cx43-pS368 / MEK / ERK signal axis.

[0039] The Cx43 Ser368 phosphorylated antigen peptide is based on the Ser368 phosphorylation site of the human Cx43 protein (UniProt ID: P17302), containing 6 amino acid residues at the N-terminus, 7 amino acid residues at the C-terminus, and Ser368 phosphorylation modification. The active amino acid sequence is SEQ ID NO:1 (specific sequence: C-PSSRASpSRASSR-COOH, where "pS" represents phosphorylation modification of serine at position 368). The Ser368 site is a PKC kinase-specific phosphorylation site, and the phosphorylation probability predicted by the NetPhos3.1 (threshold ≥0.5) and GPS6.0 (confidence ≥96%) bioinformatics models is ≥99.5%, significantly higher than other potential phosphorylation sites of the Cx43 protein (S364, S372, etc.). The corresponding non-phosphorylated antigen peptide sequence is SEQ ID NO:2 (C-PSSRASSRASSR-COOH), used for specific screening. Ser368 phosphorylated antigenic peptide with a purity ≥95% was used for immunization after being conjugated with the carrier protein KLH.

[0040] This invention also provides a method for preparing the above-mentioned specific monoclonal antibody against Cx43 Ser368 phosphorylation, comprising: Bioinformatics prediction of S0 and Cx43 phosphorylation sites: The NetPhos3.1 model (threshold ≥0.5) was used to screen potential phosphorylation sites of Cx43 protein, and the GPS6.0 model (confidence ≥96%) was used to predict PKC kinase-specific binding sites. The results showed that the phosphorylation probability of the Ser368 site was 99.59%, which is a highly specific target of the PKC family, significantly higher than other potential sites (S364: 99.36%, S372: 97.49%), thus confirming Ser368 as the core functional site.

[0041] S1. Verification of Ser368 as a functional phosphorylation site: Using Tagln-Cre-mediated VSMC-specific Cx43 KD mice (constructed using CRISPR / Cas9 technology), Cx43 KD mouse PASMCs were isolated using magnetic separation. Cell purity was verified by α-SMA immunofluorescence, and Cx43 protein and mRNA knockdown efficiency was verified by Western blot and qPCR to be ≥70%. Cx43 wild-type (WT) and Ser368 site mutation (S368A) eukaryotic expression plasmids were introduced into cells via liposome-mediated transient transfection. Samples were collected 48-72 h after transfection, and the phosphorylation function of the Ser368 site and its association with the ERK1 / 2 pathway were verified by immunoprecipitation.

[0042] In this step, Cx43 KD mice were used as the C57BL / 6J background, and the targeting sgRNA sequence was SEQ ID NO:3 (5'-GGTGCTGCTGCTGCTGCTGT-3'), which targets a key exon in the Cx43 gene coding region. PASMCs were isolated from the pulmonary artery media tissue of Cx43 KD mice and cultured to the 3rd generation for experiments. The cells in this generation showed a typical long spindle-shaped "peak-valley" morphology, an α-SMA positivity rate of ≥95%, and low basal OPN expression, confirming that they were in a normal contractile phenotype.

[0043] In this step, the specific operation of separating PASMCs by magnetic separation is as follows: After anesthetizing Cx43 KD mice, blood was removed by injecting 4℃ PBS and iron powder suspension (agarose to iron powder mass ratio 1:1, iron powder particle size 5μm) sequentially into the left ventricle. Pulmonary artery tissue was then separated under a dissecting microscope. The pulmonary artery was cut into 1.0-1.5 mm tissue blocks and digested with collagenase IV for 45 minutes. The collagenase was removed by adsorption using a magnetic separator. The cells were washed 2-3 times with M199 medium containing 20% ​​FBS, inoculated, and cultured. The medium was completely changed every 3 days. The cells were passaged when they reached 85%-90% confluence in about 7 days.

[0044] The specific transfection procedures in this step are as follows: A recombinant lentiviral vector containing the Cx43-WT or Cx43-S368A gene was constructed, co-transfected with the packaging plasmid into 293T cells, and the viral fluid was collected and concentrated to a viral titer ≥1×10⁻⁶. 8 TU / mL; Cx43 KD mouse PASMCs were inoculated into 6-well plates. When the confluence reached 60%-70%, recombinant lentivirus solution was added. The multiple of transfection (MOI) was 10-20. At the same time, 8 μg / mL polybrene was added to enhance the transfection efficiency. After culturing at 37℃ and 5% CO2 for 24 hours, the medium was replaced with fresh medium, and the culture was continued for another 48 hours. Western blot was used to verify Cx43 protein expression, confirming successful reinjection.

[0045] In this step, the specific verification procedure for validating the phosphorylation function of the Ser368 site and its association with the ERK1 / 2 pathway via immunoprecipitation is as follows: Wild-type mouse PASMCs and Cx43 KD mouse PASMCs without vector were used as controls; After culture, cells were collected, total protein and total RNA were extracted, and Cx43 mRNA expression was detected by qPCR. Western blot was used to verify that the Cx43 protein knockdown efficiency was ≥70% and the expression level was up to standard. At the same time, ERK1 / 2 phosphorylation level and expression of inflammatory factors (TNF-α, IL-1β, IL-6) were detected. Immunoprecipitation enriched Cx43 protein, and Western blot analysis with panphosphorylated serine antibody showed that the WT vector-filled group exhibited obvious phosphorylation bands, increased ERK1 / 2 phosphorylation, and upregulated expression of inflammatory factors, while the S368A vector-filled group showed no bands and no significant changes. The endogenous Cx43 phosphorylation signal in PASMCs of Cx43 KD mice without vector introduction was reduced compared with wild type, confirming that Ser368 is a functional phosphorylation site.

[0046] S2. Design and synthesize antigenic peptides: synthesize phosphorylated antigenic peptides at Cx43 Ser368 site and corresponding non-phosphorylated antigenic peptides (SEQ ID NO:2, sequence: C-PSSRASSRASSR-COOH), and conjugate them with KLH and BSA respectively to obtain immunogens and screening antigens.

[0047] S3. Animal immunization: C57BL / 6J mice were used as the immunization subjects. They were initially immunized with Freund's complete adjuvant and then given three booster immunizations with Freund's incomplete adjuvant. The serum antibody titer was ≥1:50000 7 days after the last immunization.

[0048] S4. Cell fusion and screening: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a ratio of 1:5 using PEG1500, and screened in HAT / HT medium. Antibody-specific screening was performed using four identification peptides.

[0049] In this step, the four identified peptides include: A polypeptide phosphorylated only at Ser368 site (SEQ ID NO: 1). A polypeptide without any phosphorylation sites (SEQ ID NO:2); The polypeptide phosphorylated at both Ser368 and Ser365 sites (sequence: C-PSSRpASpSRASSR-COOH). A polypeptide phosphorylated only at Ser365 (sequence: C-PSSRpASSRASSR-COOH). The screening criteria are: the antibody reacts with the first two at OD450nm ≥ 1.0 (positive), and reacts with the latter two at OD450nm < 0.5 (negative).

[0050] S5. Cloning and purification: Positive hybridoma cells were cloned using a three-round limiting dilution method. Antibodies were collected by in vitro culture in serum-free medium or by intraperitoneal injection in mice. Protein A affinity chromatography was used to purify the target antibody.

[0051] This invention also provides various applications of the specific monoclonal antibody against Cx43 Ser368 phosphorylation described herein, specifically including: 1) Application in the preparation of Cx43 Ser368 phosphorylation detection reagents; 2) Application in the preparation of formulations for detecting the dedifferentiation of PASMCs; wherein the detection is achieved by the expression of the dedifferentiation marker OPN (upregulated) and the shrinkage phenotype marker α-SMA (downregulated).

[0052] 3) Application in the preparation of drug formulations for screening drugs targeting the PKC / Cx43-pS368 signaling pathway; wherein the drug alleviates PASMC dedifferentiation and pulmonary artery remodeling by inhibiting Cx43 Ser368 phosphorylation.

[0053] This invention also provides a pulmonary hypertension diagnostic / prognostic kit, comprising the specific monoclonal antibody against Cx43Ser368 phosphorylation as described in this invention, blocking solution (5% skim milk), washing buffer (TBST), chromogenic solution (ECL), and Ser368 phosphorylated peptide standard, which can be used for dose-related risk stratification, cardiopulmonary remodeling assessment, and prognosis of nicotine-induced pulmonary hypertension.

[0054] The following are more specific embodiments to further illustrate the technical solution of the present invention: Example 1: Bioinformatics prediction of Cx43 phosphorylation sites

[0055] The NetPhos 3.1 model (threshold ≥ 0.5) was used to screen phosphorylation sites of the Cx43 protein (UniProt ID P17302) throughout its entire sequence. The results showed 38 potential phosphorylation sites identified in the 382 amino acid sequence of Cx43, including 26 serine, 6 threonine, and 6 tyrosine residues. The GPS 6.0 model (confidence ≥ 96%) was used to predict PKC kinase-specific binding sites, and the results indicated that the PKC family targets Cx43 with high confidence. Three Ser sites in the intracellular tail region: S364, S368, and S372. Among them, the S368 site had the highest prediction score (99.59%), which was significantly higher than the other sites (S364: 99.36%, S372: 97.49%). Moreover, its structural disorder (Disorderpropensity: 0.9081) and surface accessibility (ASA: 0.7685) parameters supported phosphorylation, thus identifying Ser368 as the core functional site mediated by PKC. Example 2: Construction of VSMC-specific Cx43 KD mice and magnetic isolation and culture of PASMCs

[0056] 2.1 Construction of Cx43 KD mice: Using the CRISPR / Cas9 method, Cx43 gene knockout mice [Tagln-Cre(+) vascular smooth muscle specific knockout Cx43 gene mice were constructed by hybridization of Cx43 gene mice and vascular smooth muscle specific tool mice. 2.2 Magnetic Separation of PASMCs: After anesthetizing Cx43 KD mice, 5 mL of 4℃ PBS was injected into the left ventricle to remove blood, followed by injection of a 37℃ agarose-iron powder suspension (0.05 g agarose + 0.05 g iron powder / 12 mL M199). The pulmonary artery was separated under a dissecting microscope. The pulmonary artery was cut into 1.0-1.5 mm tissue blocks, and collagenase IV solution (0.0015 g / 6 mL M199) was added. The cells were digested at 37℃ for 45 min, purified by magnetic separation, and inoculated into M199 medium containing 20% ​​FBS. The medium was completely changed after 3 days, and the cells were passaged after 7 days when they reached 90% confluence. The third generation of cells was harvested, and the purity was verified by α-SMA immunofluorescence. The expression of OPN and α-SMA was detected by immunofluorescence, confirming that the cells were in a normal contractile phenotype. The cells in this generation exhibited a typical long spindle-shaped "peak-valley" morphology. 2.3 Complementation and Validation: Lentiviral transfection and complementation of Cx43-WT and S368A vectors were performed. Western blot analysis showed that Ser368 phosphorylation, ERK1 / 2 phosphorylation, and expression of TNF-α, IL-1β, and IL-6 were significantly increased in the WT complementation group, while no significant changes were observed in the S368A group, thus validating Ser368 as a functional phosphorylation site. Example 3: Antigen peptide synthesis and conjugation, animal immunization, cell fusion and antibody purification

[0057] 3.1 Synthesis and conjugation of antigenic peptides: Phosphorylated antigenic peptides (SEQ ID NO: 1) and non-phosphorylated antigenic peptides (SEQ ID NO: 2) were synthesized in the solid phase and their purity was verified by HPLC to be ≥95%; the phosphorylated peptides were conjugated with KLH as immunogens and the non-phosphorylated peptides were conjugated with BSA as screening antigens. 3.2 Animal Immunization: C57BL / 6J mice were used as the immunization subjects. Initial immunization was performed with Freund's complete adjuvant, followed by three booster immunizations with Freund's incomplete adjuvant. Serum antibody titers were measured ≥1:64000 seven days after the final immunization. 3.3 Cell Fusion and Screening: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a 1:5 ratio using PEG1500. Cells were screened in HAT / HT medium using four identifying peptides (Ser368 phosphorylation only, no phosphorylation, Ser368+Ser365 double phosphorylation, and Ser365 phosphorylation only). The screening criteria were: OD450nm ≥1.0 for antibodies reacting with the first two peptides and <0.5 for antibodies reacting with the latter two. 3.4 Cloning and Purification: Positive hybridoma cells were cloned using a three-round limiting dilution method. Antibodies were collected after in vitro culture in serum-free medium and purified by Protein A affinity chromatography to obtain the target antibody with a purity ≥90%. Example 4: Validation of antibody specificity and dose dependence

[0058] 4.1 In vitro cell experiments: Primary PASMCs from C57BL / 6J mice were divided into a control group and a low-dose nicotine group (10⁻). 6 M), medium-dose group (10⁻ 5 M), high-dose group (10⁻ 4 After 24 hours of treatment, Cx43-pS368 expression was detected by Western blot and immunofluorescence, along with OPN and α-SMA expression. Results showed that Cx43-pS368 expression increased in a gradient with increasing nicotine dose, with the medium-dose group (10⁻⁻¹) showing the highest expression. 5 The expression of Cx43-pS368 reached its peak and was positively correlated with OPN upregulation and α-SMA downregulation, confirming that the antibody can specifically detect nicotine-induced Ser368 phosphorylation and is associated with the PASMC dedifferentiation phenotype. 4.2 In vivo animal experiments: C57BL / 6J mice were randomly divided into a control group (saline), a low-dose nicotine group (0.02 mg / kg·d), a medium-dose nicotine group (0.2 mg / kg·d), and a high-dose nicotine group (2.0 mg / kg·d). Nicotine was administered intranasally for 8 weeks to establish a dose-dependent pulmonary hypertension model. Pulmonary artery tissue and PASMCs were isolated from each group, and the expression of Cx43-pS368 was detected using the antibody of this invention. The results showed that the expression of Cx43-pS368 in the high-dose group was upregulated compared with the control group, and was positively correlated with pulmonary artery wall thickening, lumen narrowing, and increased right ventricular systolic pressure, confirming that the antibody can quantify the association between in vivo nicotine exposure dose and phosphorylation signal. Example 5: Application of antibodies in drug screening for the PKC / Cx43-pS368 signaling pathway

[0059] 5.1 Experimental Materials: Nicotine-induced PASMCs model (10⁻ 5 5.2 Experimental Procedure: PASMCs were divided into a control group, a nicotine group, and a nicotine + chelidonine pretreatment group (pretreatment for 1 hour). Western blot and immunofluorescence methods were used to detect Cx43-pS368 expression using the antibody of this invention, along with OPN and α-SMA. EdU assay was used to detect cell proliferation, and scratch assay was used to detect cell migration. (Note: The text also mentions nicotine treatment for 24 hours, PKC inhibitor chelidonine (concentration 100-500 nM), and the Cx43-pS368-specific antibody prepared in this invention, but these appear unrelated to the main topic and are likely separate data points.) 5.3 Results: In the nicotine group, Cx43-pS368 expression was significantly increased, OPN was upregulated, and α-SMA was downregulated, resulting in significantly increased cell proliferation and migration rates. In the chelidonine pretreatment group, Cx43-pS368 expression was decreased, while OPN and α-SMA expression recovered to levels close to the control group. Cell proliferation and migration were significantly inhibited, confirming that the antibody can specifically detect PKC-mediated Ser368 phosphorylation and can be used for drug screening targeting this signaling pathway. Example 6: Validation of Antibody Application Effects: Preparation of a Pulmonary Hypertension Diagnostic / Prognostic Kit

[0060] The kit contains: a specific monoclonal antibody against Cx43 Ser368 phosphorylation as described in this invention, blocking buffer (5% skim milk), wash buffer (TBST), chromogenic solution (ECL), Ser368 phosphorylated peptide standard, ELISA plate, and HRP-conjugated secondary antibody. In use, the expression level of Cx43-pS368 in clinical samples (pulmonary artery tissue or PASMC) is detected by ELISA. Combined with nicotine exposure history, dose-related risk stratification, cardiopulmonary remodeling assessment, and prognostic prediction for nicotine-induced pulmonary hypertension can be achieved.

[0061] The sequence list involved in the above embodiments is shown in the following table: Sequence Number Sequence Type Sequence Length Sequence Content Sequence Source SEQ ID NO: 1 Phosphorylated Antigenic Peptide 13 amino acids C-PSSRASpSRASSR-COOH Synthetic SEQ ID NO: 2 Non-phosphorylated Antigenic Peptide 13 amino acids C-PSSRASSRASSR-COOH Synthetic The following is an explanation of the various figures involved in this embodiment: Figure 1A procedure for identifying VSMC-specific Cx43 KD mice and magnetically separating PASMCs was developed. This demonstrates the ability to rapidly separate high-purity mouse PASMCs. The images show: (a) C57BL / 6J mice with limbs fixed after anesthesia; (b) mouse fur opened; (c) cardiopulmonary tissue before perfusion; (d) lung tissue after perfusion; (e) lung tissue after agarose iron powder perfusion; (f) lung lobes separated in a laminar flow hood; (g) separated lung lobes; and (h) lung tissue 1 h after collagenase digestion. Figure 1 This indicates that high-purity mouse PASMCs can be rapidly isolated.

[0062] Figure 2 Immunofluorescence identification of α-SMA in PASMCs. This indicates that the cells are PASMCs. In the figures: a: Blue represents the cell nucleus (×200). b: Green represents α-SMA (a marker of PASMCs) (×200). c: Overlay of Figure a and Figure b. Figure 2 This indicates that the cells are PASMCs.

[0063] Figure 3 This image shows the colocalization of Cx43-pS368 with dedifferentiation markers under nicotine intervention. It indicates that nicotine intervention can induce phosphorylation of the Cx43 Ser368 site in mouse PASMCs and increase the degree of dedifferentiation in PASMCs. (A) Representative immunofluorescence images of OPN and Cx43-pS368 expression in pulmonary arteries. (B, C) Quantitative analysis of OPN and Cx43-pS368 fluorescence intensity, n=5. (D) Representative immunofluorescence images of OPN and Cx43-pS368 expression in pulmonary artery smooth muscle cells (PASMCs). (E, F) Quantitative analysis of OPN and Cx43-pS368 fluorescence intensity, n=5. (G, H) Representative Western blot images and statistical graphs of OPN and Cx43-pS368 protein expression in nicotine-incubated PASMCs, n=3. Data are expressed as mean ± standard error. *P<0.05; **P<0.01. Figure 3 This indicates that nicotine intervention can induce phosphorylation at the Cx43 Ser368 site of mouse PASMCs and increase the degree of dedifferentiation of PASMCs.

[0064] Figure 4 Correlation analysis of the right ventricular hypertrophy index showed that nicotine intervention can induce right ventricular hypertrophy in mice. Figure 4 This indicates that nicotine intervention can cause right ventricular hypertrophy in mice.

[0065] Figure 5Bioinformatics predictions for Cx43 phosphorylation sites (NetPhos 3.1 GPS 6.0) show that S368 is the most likely core site for the PKC kinase family to regulate Cx43 phosphorylation. The NetPhos 3.1 machine learning model was used to predict individual phosphorylation sites in Cx43. A prediction score higher than 0.5 (purple bar) indicates the presence of a phosphorylation site. The NetPhos 3.1 model identified all potential phosphorylation sites in the Cx43 sequence and determined the protein kinases responsible for activating these phosphorylation events. The GPS 6.0 model specifically highlighted the sites in the Cx43 sequence activated by the PKC protein kinase family (i.e., S364, S368, and S372), with scores of 99.36%, 99.59%, and 97.49%, respectively, all exceeding the critical value of 96.19%. The classic GPS 6.0 model predicted the disorder tendency and ASA score of these three sites in the Cx43 sequence activated by the PKC protein kinase family. Figure 5 This indicates that S368 is the most likely core site for the PKC kinase family to regulate Cx43 phosphorylation.

[0066] Figure 6 The study investigated the reversal effects of PKC inhibitors on Cx43-pS368 expression and the dedifferentiation, proliferation, and migration of PASMCs. This indicated that the PKC inhibitor (chelerythrine) blocked the nicotine-induced dedifferentiation of PASMCs by inhibiting Cx43 Ser368 phosphorylation. The study also examined the effects of Chelerythrine-Cl on the dedifferentiation of PASMCs and Cx43-pS368 expression in C57BL / 6J mice. PASMCs were pretreated with 500 nM chelerythrine-Cl for 1 hour before nicotine exposure. (A) Representative images of Edu-stained PASMCs and (B) Quantitative analysis of Edu-positive cells, scale bar = 20 μm, n = 5. (C) Representative images of PASMCs obtained from the scratch assay and (D) Quantitative results, scale bar = 20 μm, n = 5. (E) Immunofluorescence staining images and quantitative analysis of Cx43-pS368 and OPN expression in PASMCs; (F, G) Quantitative analysis of Cx43-pS368 and OPN in PASMCs, scale bar = 20 μm, n = 5. (H) Representative Western blot images and quantitative analysis of OPN expression in PASMCs; (I) Quantitative analysis of Cx43-pS368 expression in PASMCs, n = 3. Data are expressed as mean ± standard error. * P <0.05;** P <0.01. Figure 6 This indicates that the PKC inhibitor (chelidonine) blocked the promoting effect of nicotine on the dedifferentiation of PASMCs by inhibiting Cx43 Ser368 phosphorylation.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A specific monoclonal antibody against phosphorylation at the Ser368 site of Cx43, characterized in that, It is prepared by immunizing animals with phosphorylated antigenic peptides at the Cx43Ser368 site. It can specifically bind to the phosphorylated form of Cx43Ser368 in PASMCs in pulmonary hypertension specimens, without cross-reacting with non-phosphorylated Cx43 or other phosphorylated forms. The equilibrium dissociation constant KD≤100nM. It can detect nicotine exposure-induced phosphorylation signals in a dose-dependent manner and correlate the proliferation, migration, dedifferentiation and cardiopulmonary remodeling of PASMCs through the PKC / Cx43-pS368 / MEK / ERK signal axis.

2. The specific monoclonal antibody against phosphorylation at Cx43 Ser368 site according to claim 1, characterized in that: The Cx43 Ser368 phosphorylated antigenic peptide is based on the Ser368 phosphorylation site of the human Cx43 protein, and includes 6 amino acid residues on the N-terminal side, 7 amino acid residues on the C-terminal side, and Ser368 phosphorylation modification. The active amino acid sequence is SEQ ID NO:

1. The Ser368 site is a PKC kinase-specific phosphorylation site. According to the NetPhos3.1 and GPS6.0 bioinformatics models, the phosphorylation probability is ≥99.5%, which is significantly higher than other potential phosphorylation sites of the Cx43 protein. The phosphorylated antigenic peptide at the Cx43 Ser368 site has a purity ≥95% and is used for immunization after being conjugated with the carrier protein KLH.

3. The method for preparing the specific monoclonal antibody against Cx43 Ser368 phosphorylation as described in claim 2, characterized in that, include: Bioinformatics prediction of S0 and Cx43 phosphorylation sites: The NetPhos3.1 model was used to screen potential phosphorylation sites of Cx43 protein, and the GPS6.0 model was used to predict PKC kinase-specific binding sites. The results showed that the phosphorylation probability of the Ser368 site was 99.59%, which is a highly specific target of the PKC family, significantly higher than other potential sites, thus confirming Ser368 as the core functional site. S1. Verification of Ser368 as a functional phosphorylation site: Using Tagln-Cre-mediated VSMC-specific Cx43KD mice, PASMCs were isolated using magnetic separation. Cell purity was verified by α-SMA immunofluorescence. Western blot and qPCR confirmed that the knockdown efficiency of Cx43 protein and mRNA was ≥70%. Cx43 wild-type and Ser368 site mutant eukaryotic expression plasmids were introduced into cells via liposome-mediated transient transfection. Samples were collected 48-72 h after transfection, and the phosphorylation function of the Cx43 Ser368 site and its association with the ERK1 / 2 pathway were verified by immunoprecipitation. S2. Design and synthesize antigenic peptides: synthesize phosphorylated antigenic peptides at Cx43 Ser368 site and corresponding non-phosphorylated antigenic peptides, and conjugate them with KLH and BSA respectively to obtain immunogens and screening antigens. S3. Animal immunization: C57BL / 6J mice were used as the immunization subjects. They were initially immunized with Freund's complete adjuvant and then given three booster immunizations with Freund's incomplete adjuvant. The serum antibody titer was ≥1:50000 seven days after the last immunization. S4. Cell fusion and screening: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a ratio of 1:5 using PEG1500, and screened in HAT / HT medium. Antibody specificity screening was performed using four identification peptides. S5. Cloning and Purification: Positive hybridoma cells were cloned using a three-round limiting dilution method. Antibodies were collected by in vitro culture in serum-free medium or by intraperitoneal injection in mice. Protein A affinity chromatography was used for purification to obtain the target antibody.

4. The method for preparing the specific monoclonal antibody against Cx43 Ser368 phosphorylation according to claim 3, characterized in that: In step S1, Cx43 KD mice were used as the C57BL / 6J background, and the targeting sgRNA sequence was SEQ ID NO:3, targeting a key exon in the coding region of the Cx43 gene. PASMCs were isolated from the pulmonary artery media tissue of Cx43 KD mice and cultured in primary culture to the 3rd generation for experiments. The cells in this generation showed a typical long spindle-shaped "peak-valley" morphology, an α-SMA positivity rate of ≥95%, and low basal OPN expression, confirming that they were in a normal contractile phenotype. In step S1, the specific operation of separating PASMCs by magnetic separation is as follows: After anesthetizing the mice, the left ventricle was sequentially injected with 4°C PBS to remove blood and 37°C agarose iron powder suspension, and the pulmonary artery tissue was separated under a dissecting microscope. The pulmonary artery was cut into 1.0-1.5 mm tissue blocks and digested with collagenase IV for 45 minutes. The collagenase was removed by adsorption using a magnetic separator. The cells were washed 2-3 times with M199 medium containing 20% ​​FBS, inoculated and cultured, and the medium was completely changed every 3 days. The cells were passaged when they reached 85%-90% confluence in about 7 days. In step S1, the specific transfection operations are as follows: A recombinant lentiviral vector containing the Cx43-WT or Cx43-S368A gene was constructed, co-transfected with the packaging plasmid into 293T cells, and the viral fluid was collected and concentrated to a viral titer ≥1×10⁻⁶. 8 TU / mL; Cx43 KD mouse PASMCs were inoculated into 6-well plates. When the confluence reached 60%-70%, recombinant lentivirus solution was added. The multiple of transfection (MOI) was 10-20. At the same time, 8 μg / mL polybrene was added to enhance the transfection efficiency. After culturing at 37℃ and 5% CO2 for 24 hours, the medium was replaced with fresh medium, and the culture was continued for 48 hours. Western blot was used to verify Cx43 protein expression, confirming successful reinjection. In step S1, the specific verification procedure for verifying the phosphorylation function of the Cx43 Ser368 site and its association with the ERK1 / 2 pathway via immunoprecipitation is as follows: Wild-type mouse PASMCs and Cx43 KD mouse PASMCs without vector were used as controls; After culture, cells were collected, total protein and total RNA were extracted, and Cx43 mRNA expression was detected by qPCR. Western blot was used to verify that the Cx43 protein knockdown efficiency was ≥70% and the expression level was up to standard. At the same time, ERK1 / 2 phosphorylation level and inflammatory factor expression were detected. Immunoprecipitation enriched Cx43 protein, and Western blot analysis with panphosphorylated serine antibody showed that the WT vector-filled group exhibited a significant phosphorylation band, increased ERK1 / 2 phosphorylation, and upregulated expression of inflammatory factors, while the S368A vector-filled group showed no band and no significant changes. The endogenous Cx43 phosphorylation signal in PASMCs of Cx43 KD mice without vector introduction was reduced compared with wild type, verifying that the Cx43Ser368 site is a functional phosphorylation site.

5. The method for preparing the specific monoclonal antibody against Cx43 Ser368 phosphorylation according to claim 3, characterized in that: In step S4, the four identified polypeptides include: Peptides phosphorylated only at the Cx43 Ser368 site; Peptides without any phosphorylation sites; A polypeptide phosphorylated at both Ser368 and Ser365 sites of Cx43; A peptide phosphorylated only at the Cx43 Ser365 site; The screening criteria were: the antibody reacted with the first two antibodies at an OD450nm ≥ 1.0, and reacted with the latter two antibodies at an OD450nm < 0.

5.

6. The use of the specific monoclonal antibody against Cx43 Ser368 phosphorylation as described in claim 1 or 2 in the preparation of a Cx43Ser368 phosphorylation detection reagent.

7. The use of the specific monoclonal antibody against Cx43 Ser368 phosphorylation as described in claim 1 or 2 in the preparation of a formulation for detecting PASMC dedifferentiation; wherein, The detection was achieved by associating the expression of the dedifferentiation marker OPN and the shrinkage phenotype marker α-SMA.

8. The use of the specific monoclonal antibody against Cx43 Ser368 phosphorylation as described in claim 1 or 2 in the preparation of drug formulations for screening the PKC / Cx43-pS368 signaling pathway; wherein, The drug alleviates PASMC dedifferentiation and pulmonary artery remodeling by inhibiting Cx43Ser368 phosphorylation.

9. A diagnostic / prognostic kit for pulmonary hypertension, characterized in that, The mixture comprises a specific monoclonal antibody against Cx43Ser368 phosphorylation as described in claim 1 or 2, a blocking buffer, a washing buffer, a chromogenic solution, and a Cx43Ser368 phosphorylated peptide standard.

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