阅读说明：本技术 一种抗cd105特异性单克隆抗体及其应用 (anti-CD 105 specific monoclonal antibody and application thereof ) 是由 彭劲武 李尚彧 闵豆 程育苗 于 2021-01-21 设计创作，主要内容包括：本发明涉及一种抗CD105特异性单克隆抗体,其重链可变区具有如SEQ ID No：6所示的氨基酸序列,其轻链可变区具有如SEQ ID No：7所示的氨基酸序列。该抗CD105特异性单克隆抗体,编码所述抗CD105特异性单克隆抗体的重链可变区的核苷酸序列如SEQ ID No：4所示,编码所述抗CD105特异性单克隆抗体的轻链可变区的核苷酸序列如SEQ ID No：5所示。本发明的CD105抗体与人CD105蛋白的结合具有特异性强、检测灵敏度高的特点。本发明抗CD105特异性单克隆抗体适合用于各种恶性肿瘤的诊断检测中,可提高检测的准确度。(The invention relates to an anti-CD 105 specific monoclonal antibody, wherein a heavy chain variable region of the monoclonal antibody has a sequence shown in SEQ ID No: 6, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 7. The nucleotide sequence of the heavy chain variable region of the anti-CD 105 specific monoclonal antibody is shown as SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 105 specificity is shown as SEQ ID No: 5, respectively. The combination of the CD105 antibody and the human CD105 protein has the characteristics of strong specificity and high detection sensitivity. The anti-CD 105 specific monoclonal antibody is suitable for diagnosis and detection of various malignant tumors, and can improve the detection accuracy.)

1. An anti-CD 105 specific monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence as shown in SEQ ID No: 6, and the light chain variable region has an amino acid sequence shown as SEQ ID No: 7.

2. An anti-CD 105 specific monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region encoding the anti-CD 105 specific monoclonal antibody is shown in SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 105 specificity is shown as SEQ ID No: 5, respectively.

3. The anti-CD 105 specific monoclonal antibody according to claim 1 or 2, wherein the anti-CD 105 monoclonal antibody is a mouse IgG1 subtype monoclonal antibody.

4. A method for preparing an anti-CD 105 specific monoclonal antibody, comprising the steps of:

s1 immunization: using SEQ ID No:1-3 as immunogen, coupling via KLH or OVA, immunizing different Balb/c mice, taking blood to measure serum antibody titer, and selecting mice with highest antibody titer corresponding to each synthetic polypeptide for cell fusion;

s2 cell fusion: the myeloma cells are sp2/0 mouse myeloma-like cells of mouse origin; taking mouse spleen lymphocytes and sp2/0 mouse myeloma-like cells for cell fusion, and culturing in HAT culture medium to obtain hybridoma cells;

s3, screening: selecting cell clones within 7-10 days of fusion, performing ELISA test by using CD105 antigen, performing immunohistochemical detection by using CD105 positive tissues, performing limited dilution on positive cells, performing ELISA test and IHC test, and selecting monoclonal stable strains which are positive and have high positive values from all diluted samples, namely positive hybridoma cell strains;

s4 preparation of antibody: preparing mouse ascites by using the cell line of the positive hybridoma cell strain, collecting the ascites, and purifying the ascites by adopting affinity chromatography to obtain an anti-CD 105 specific monoclonal antibody; alternatively, the first and second electrodes may be,

and (3) culturing and expanding the positive hybridoma cell strain by using a serum DMEM medium, centrifuging at a low speed, removing a supernatant, transferring cells to a serum-free medium, continuously culturing for 1-2 weeks, collecting cell suspension, centrifuging, taking the supernatant, and purifying by using an affinity chromatography method to obtain the anti-CD 105 specific monoclonal antibody.

5. A method for preparing an anti-CD 105 specific monoclonal antibody, which is characterized in that the amino acid sequence of the heavy chain variable region of the anti-CD 105 specific monoclonal antibody is shown as SEQ ID No: 6, the amino acid sequence of the light chain variable region of the anti-CD 105 specific monoclonal antibody SEQ ID No: 7, constructing an expression vector of the CD105 antibody in vitro, performing amplification extraction on the expression vector through escherichia coli, removing endotoxin, transfecting Expi-293F cells, continuing culturing after transfection, collecting cell supernatant, and purifying by adopting an affinity chromatography to obtain the anti-CD 105 specific monoclonal antibody.

6. A kit or an antibody chip for detecting CD105 protein, comprising the anti-CD 105 specific monoclonal antibody according to any one of claims 1 to 3 or the anti-CD 105 specific monoclonal antibody prepared by the preparation method according to any one of claims 4 to 5.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-CD 105 specific monoclonal antibody and application thereof.

Background

CD105, also known as Endoglin, is a homodimeric transmembrane glycoprotein present on cell surfaces and capable of specifically binding transforming growth factor beta (TGF-beta), a polypeptide cytokine with angiogenic activity, and promoting tumorigenesis by modulating angiogenesis, immune response and EMT. The generation of tumor new vessels plays an important role in tumor proliferation and metastasis, and CD105, which is one of the markers of vascular endothelial cell proliferation, can directly participate in angiogenesis and plays an important role in the growth process of tumor vessels.

The research shows that CD105 has close relationship with tumor tissues such as lung adenocarcinoma, renal cell carcinoma, colorectal cancer, breast cancer, cervical cancer and the like, and the CD105 is strongly expressed on malignant tumor tissues of different sources and peripheral vascular endothelial cells thereof, mainly expressed in immature blood vessels and not expressed on normal tissue blood vessels. Therefore, the method can be used for researching angiogenesis in various malignant tumor tissues and can also be used as a detection index for growth, metastasis and prognosis of various malignant tumors.

The CD 105-based detection index is important to be applied to various malignant tumors, metastasis, prognosis and the like, so that the method has important significance for obtaining the anti-CD 105 antibody with high sensitivity and strong specificity.

Disclosure of Invention

Technical problem to be solved

In view of the problems of the prior art, the present invention provides an anti-CD 105 specific monoclonal antibody, which can be widely applied to diagnosis of various malignant tumors and has extremely high specificity and sensitivity.

The CD 105-resistant specific monoclonal antibody provided by the invention is produced by a mouse hybridoma cell strain, and is found by immunohistochemical detection of various tumor tissues, so that the antibody can well identify the occurrence and metastasis conditions of malignant tumors, and can be used for immunological diagnosis of the malignant tumors.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the present invention provides an anti-CD 105 specific monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence as shown in SEQ ID No: 6, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 7.

In a second aspect, the present invention provides an anti-CD 105 specific monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region encoding the anti-CD 105 specific monoclonal antibody is as shown in SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 105 specificity is shown as SEQ ID No: 5, respectively.

Wherein the subclass of the anti-CD 105 monoclonal antibody is mouse IgG1 type monoclonal antibody measured by ELISA technology, and the affinity constant is 8 × 10^-7(mol/L). Immunohistochemical experiments show that the antibody can specifically recognize CD105 protein, including but not limited to recombinant CD105 antigen protein, CD105 molecule in lymphohematopoietic system, and the like.

In a third aspect, the present invention provides a method for preparing an anti-CD 105 monoclonal antibody, comprising:

s1 immunization: using SEQ ID No:1-3 as immunogen, coupling via KLH or OVA, immunizing different Balb/c mice, taking blood to measure serum antibody titer, and selecting mice with highest antibody titer corresponding to each synthetic polypeptide for cell fusion;

s2 cell fusion: the myeloma cells are sp2/0 mouse myeloma-like cells of mouse origin; taking mouse spleen lymphocytes and sp2/0 mouse myeloma-like cells for cell fusion, and culturing in HAT culture medium to obtain hybridoma cells;

s3, screening: selecting cell clones within 7-10 days of fusion, performing ELISA test by using CD105 antigen, performing immunohistochemical detection by using CD105 positive tissues, performing limited dilution on positive cells, performing ELISA test and IHC test, and selecting a monoclonal stable strain which is a positive hybridoma cell strain 813A5D5 and all diluted samples are positive and have high positive values;

s4 preparation of antibody: preparing mouse ascites by using a cell line of a positive hybridoma cell strain 813A5D5, collecting the ascites, and purifying the ascites by using Protein A/G column affinity chromatography to obtain an anti-CD 105 specific monoclonal antibody; alternatively, the first and second electrodes may be,

and (3) culturing and expanding the positive hybridoma cell strain 813A5D5 by using a serum DMEM medium, centrifuging at a low speed, removing a supernatant, transferring cells to a serum-free medium, continuously culturing for 1-2 weeks, collecting a cell suspension, centrifuging, taking the supernatant, and purifying by using an affinity chromatography method to obtain the anti-CD 105 specific monoclonal antibody.

The anti-CD 105 monoclonal antibody was secreted by a cell line of mouse hybridoma 813A5D 5.

The preparation method also comprises the steps of monoclonal antibody subtype identification and affinity constant determination: the obtained anti-CD 105 specific monoclonal antibody subtype is determined to be IgG1 type monoclonal antibody by ELISA technology, and the affinity constant is 8 multiplied by 10^-7(mol/L)。

The result of immunohistochemical experimental detection shows that the antibody has the function of specifically recognizing CD105 protein molecules in malignant tumor tissues.

Preferably, the synthetic polypeptide used as immunogen in S1 is the amino acid sequence shown in SEQ ID NO 1-3, and the sequence of SEQ ID NO 1 is DRGTLPLAVAL LLA; 2, IFPEKNIRGFKLPDTP as shown in SEQ ID NO; SEQ ID NO 3 has the sequence NHSIGSTQSTPCSTS.

In a fourth aspect, the present invention provides a method for preparing an anti-CD 105 specific monoclonal antibody, wherein the heavy chain variable region of the anti-CD 105 specific monoclonal antibody has the amino acid sequence of SEQ ID No: 6, the amino acid sequence of the light chain variable region SEQ ID No: 7, constructing an expression vector of the CD105 antibody in vitro, performing amplification extraction on the expression vector through escherichia coli, removing endotoxin, transfecting Expi-293F cells, continuing culturing after transfection, collecting cell supernatant, and purifying by adopting an affinity chromatography to obtain the anti-CD 105 specific monoclonal antibody.

In a fifth aspect, the present invention provides a kit or an antibody chip for detecting CD105 protein, comprising the anti-CD 105 specific monoclonal antibody according to any one of the above embodiments.

The anti-CD 105 monoclonal antibody is used for detecting the expression condition of CD105 in malignant tumor tissues and normal tissue cells, and the detection method comprises one or more of the following steps: immunohistochemistry, immunoblotting, and enzyme-linked adsorption assays.

Preferably, the anti-CD 105 monoclonal antibody is used in an immunohistochemical pathological diagnostic agent.

(III) advantageous effects

The anti-CD 105 monoclonal antibody obtained by the invention is secreted by a hybridoma 813A5D5 cell line and is an IgG1 antibody. The anti-CD 105 monoclonal antibody obtained by the invention has strong specificity and high sensitivity in combination with the CD105 protein, and can accurately identify the expression of CD105 in malignant tumors. The antibody can be applied to detection processes of Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chips and the like.

Drawings

FIG. 1 is a graph showing the results of Western Blot detection of monoclonal antibodies in lysates of CD105 positive tissues, namely H.ovary (human ovarian cancer cell line) and H.placenta (human placental tissue), wherein the primary antibody is anti-CD 105 monoclonal antibody 813A5D5 prepared in the example, and used at a concentration of 2. mu.g/mL.

FIG. 2 is a graph comparing the representative results of immunohistochemical detection (positive staining signals) of CD105 monoclonal antibody 813A5D5 prepared in the present example and commercially available CD105 (currently most commonly used commercial CD105 antibody) monoclonal antibody on lung adenocarcinoma tissue samples, with the primary antibody being used at a concentration of 1. mu.g/mL.

FIG. 3 is a graph comparing the results of immunohistochemical detection (positive staining signals) of CD105 monoclonal antibody 813A5D5 prepared in the example of the present invention and commercially available CD105 (currently most commonly used commercial CD105 antibody) monoclonal antibody on breast cancer tissue samples, and the concentration of primary antibody used was 1. mu.g/mL.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

The CD105 protein molecule is analyzed according to a published sequence, and according to the structure on a cell membrane, the antigenicity, the hydrophilicity and hydrophobicity of the constituent amino acids and the secondary structure, the CD105 protein molecule is selected from SEQ ID No:1-3 as immunogen to immunize Balb/c mouse. The positive hybridoma cell line 813A5D5 capable of efficiently secreting monoclonal antibodies is obtained by fusing, screening and cloning with the mouse myeloma-like cell. The hybridoma cell line is used for preparing mouse ascites, and Protein A/G column affinity chromatography is used for purifying the ascites to obtain the anti-CD 105 monoclonal antibody (or DMEM culture medium of serum is adopted for culture and expansion, then serum-free culture medium is adopted for culture, centrifugation is carried out to obtain supernatant, affinity chromatography is used for purification, and then the anti-CD 105 monoclonal antibody is prepared). The subclass of the monoclonal antibody is IgG1 type monoclonal antibody determined by ELISA technique, and the affinity constant is 8 × 10^-7(mol/L). Immunohistochemical experiments show that the antibody can specifically recognize CD105 protein, including but not limited to recombinant CD105 antigen protein and CD105 molecule in lymphohematopoietic system.

The following explains the embodiments of the present invention with reference to specific examples.

Example 1

Preparation of hybridoma cell line 813A5D5 secreting anti-CD 105 specific monoclonal antibody:

(1) the following amino acid sequences of the synthetic polypeptide are obtained by chemical synthesis:

SEQ ID NO 1：DRGTLPLAVAL LLA；

SEQ ID NO 2：IFPEKNIRGFKLPDTP；

SEQ ID NO 3：NHSIGSTQST PCSTS。

(2) mouse immunization: the polypeptides with the sequences shown in SEQ ID NO 1-3 are respectively mixed and emulsified with Freund's complete adjuvant 1:1, different BALB/c mice are respectively immunized by adopting a subcutaneous injection method, and after two weeks, the polypeptides are mixed with Freund's incomplete adjuvant 1:1 for secondary immunization. After twice immunization, blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; and determining whether to boost the immunity according to the result. Detecting the serum titer of the mice to reach 1:100000, then performing abdominal cavity boosting immunization (30 mu g) by using the polypeptide (without adjuvant), cutting tail and collecting blood after 3 days, detecting the serum titer, selecting the mice with the highest antibody titer corresponding to the antigen immunization of SEQ ID No:1-3, and preparing for cell fusion.

(3) Hybridoma cell fusion: the myeloma cells were derived from mouse sp2/0 mouse myeloma-like cell line and were in logarithmic growth phase at the time of fusion. Taking the spleen of the immunized mouse to prepare lymphocyte single cell suspension. Mixing splenocytes of immunized mouse and SP2/0 mouse myeloma-like cells at a ratio of 4:1 in a 50mL centrifuge tube, adding 50% PEG1500 dropwise, adding incomplete culture medium and the rest stop solution, centrifuging, removing supernatant, adding HAT culture medium (HAT (H-Hypoxanthine Hypoxanthine, A-Aminopterin, T-thymine Thymidine) selective culture medium), suspending, mixing, metering MC to 50mL, packaging in 96-well plate, and standing at 37 deg.C with 5% CO₂Culturing in a constant temperature incubator.

(4) Screening and cloning: cell clones were picked at 8 days of fusion and tested by ELISA using CD105 antigen. The cell line number was labeled. The supernatants of the ELISA positive cell lines were subjected to immunohistochemical detection (IHC detection). ELISA and IHC positive well cells were diluted in a limiting manner and cultured in 96-well plates. Each well was 100. mu.L, and the cells were cultured in an incubator. Cell supernatants were identified until the full plate results of the 96-well plate were positive by ELISA assay. All the well plates were selected as positive monoclonal stable strains with high positive values, and the hybridoma cell 1 strain secreting the specific monoclonal antibody, 813A5D5, was obtained.

Example 2

The monoclonal antibody is prepared and purified by adopting the following two modes:

the first method is as follows: cell line 813A5D5 was expanded to approximately 4X 10 by culturing in 10cm dishes in DMEM medium containing 15% serum⁷At one time, centrifuge at 1200rpm for 6 minutes, discard the supernatantAnd the cells were transferred to 2L spinner flasks and serum-free medium was added. And after continuing culturing for 2 weeks, collecting cell suspension, centrifuging, taking supernatant, purifying the supernatant by affinity chromatography, selecting corresponding column materials according to antibody subtypes, wherein the monoclonal antibody 813A5D5 subtype is IgG1, and purifying by adopting protein G. Measuring the concentration of the purified monoclonal antibody, subpackaging (100 μ L/tube, concentration of 1mg/mL), and storing in a refrigerator at 4-8 deg.C.

Injecting liquid paraffin into the abdominal cavity of the selected mouse to stimulate the immune response of the mouse; inoculating the cell strain 813A5D5 into the abdominal cavity of a mouse after one week, extracting ascites after one week, placing the extracted ascites into a centrifuge tube, balancing on a balance, and then placing into a high-speed refrigerated centrifuge for centrifugation; after centrifugation is finished, removing upper-layer grease and sediments with blood filaments at the bottom, filtering the residual liquid with gauze, adding saturated ammonium sulfate overnight, taking the precipitate, redissolving with PBS, purifying by affinity chromatography, selecting corresponding column materials according to antibody subtype, wherein the monoclonal antibody 813A5D5 subtype is IgG1, and purifying by protein G. The purified monoclonal antibody was assayed for concentration, dispensed (100. mu.L/tube, concentration 1mg/mL) and stored at 4-8 ℃.

For convenience of description (to distinguish from the existing anti-CD 105 monoclonal antibody), the anti-CD 105 specific monoclonal antibody secreted from the hybridoma cells is hereinafter abbreviated as 813A5D5 anti-CD 105 monoclonal antibody or 813A5D5 anti-CD 105 monoclonal antibody.

The 813A5D5 anti-CD 105 monoclonal antibody prepared in the two ways described above was now sequenced:

separating total RNA from hybridoma cells according to the instruction of a reagent TriZol, carrying out reverse transcription on the total RNA into cDNA according to the instruction of a TIANCcript first strand cDNA synthesis kit, amplifying antibody fragments of VH, VL, CH and CL according to specific primers, respectively cloning the amplified antibody fragments into a standard cloning vector, and sequencing.

The sequencing result is as follows: 813A5D5 the heavy chain variable region amino acid sequence of the anti-CD 105 monoclonal antibody is represented by SEQ ID No: 4, and the light chain variable region amino acid sequence of the anti-CD 105 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No: 5 is encoded by the DNA sequence shown in the specification. Correspondingly, the heavy chain variable region amino acid sequence of the anti-CD 105 monoclonal antibody is SEQ ID No: 6, and the light chain variable region amino acid sequence of the CD105 monoclonal antibody is SEQ ID No: 7.

Example 3

The 813A5D5 antibody was detected as a subtype by ELISA. The results showed that the subtype of the 813A5D5 antibody was IgG 1.

Example 4

Cell lysates of h.ovary human ovarian cancer cells and human placental tissue h.placenta were prepared, and Western Blot detection was performed by adding 813A5D5 anti-CD 105 monoclonal antibody prepared in example 2 (manner one). The results are shown in fig. 1, and it can be seen from the results in fig. 1 that a band of CD105 protein was clearly detected in both h.ovary and h.planta cell lysates.

Example 5

Immunohistochemical detection of lung adenocarcinoma tissue samples using 813A5D5 anti-CD 105 monoclonal antibody as the primary antibody. The method comprises the following steps:

(1) sample preparation: the formalin fixed paraffin embedded section is baked for 1 to 2 hours in an oven at the temperature of 60 ℃ and stored at room temperature for later use.

(2) Dewaxing and hydrating: paraffin slices are soaked in fresh dimethylbenzene for 2 times for dewaxing, and each time is 10 min. Then sequentially soaking in anhydrous ethanol (2 times, 3min each time), 95% ethanol (3min), 85% ethanol (3min), and 70% ethanol for 5min for hydration, washing with purified water for 2 times, 3min each time.

(3) Antigen retrieval: repairing with high temperature heat repairing method for 3min (98 deg.C for 20min if using automatic repairing instrument), naturally cooling to room temperature, looping the tissue to be tested (lung adenocarcinoma tissue in this embodiment) with immunohistochemical pen, washing with purified water for 2 times, 3min each time.

(4) Inactivation of endogenous peroxidase: dropping appropriate amount of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 10min, washing with purified water for 2 times (3min each time), and washing with PBST for 1 time.

(5) Primary and blank control antibody incubations: 2 drops of 813A5D5 anti-CD 105 monoclonal antibody at equal concentration and a control commercial CD105 monoclonal antibody were added to completely cover the tissue, incubated in a 37 ℃ incubator for 1h, and washed 3 times with PBST (phosphate buffered saline containing Tween-20) for 5min each.

(6) And (3) secondary antibody incubation: and (4) performing secondary antibody incubation according to the instruction of the DAB staining solution kit of the secondary antibody staining system, after the incubation is finished, washing the PBST by using the washing sheet for 3 times, 5min each time, and washing by using purified water for 1 time.

(7) DAB color development: preparing DAB color developing solution according to the DAB staining solution kit specification, dripping a proper amount of the prepared DAB color developing solution until the tissue is completely covered, stopping staining when the color is not deepened, and washing with purified water for 3 times.

(8) Hematoxylin lining staining: the sections were counterstained according to the protocol and recommendations of the hematoxylin manufacturer's instructions, washed back to blue with PBST or tap water.

(9) And (3) dehydrating and transparency: soaking in 70%, 85%, 95%, 100%, 100% gradient alcohol for 3min each time; 2 times, 5min each time, xylene was clear.

(10) Sealing: the samples were mounted with neutral gum.

The above-prepared patches were observed by fluorescence microscopy, and as shown in fig. 2, CD105 protein was expressed in a specific cell membrane in lung adenocarcinoma tissue, and the detection signal of the anti-CD 105 monoclonal antibody of 813A5D5 clone was significantly stronger than that of the commercially available CD105 group. Under the same antibody concentration, the staining positive signal of the 813A5D5 anti-CD 105 monoclonal antibody is obviously enhanced compared with the staining positive signal of a commercially available CD105 group, which indicates that the antibody has higher sensitivity, lower antibody concentration can be used in product design, the production cost is reduced, and the background is reduced under the condition of ensuring the sensitivity. The method facilitates the judgment of pathologists according to the CD105 detection result and experience, and is more accurate for detecting and distinguishing immune tissues or immune related diseases.

Example 6

Immunohistochemical detection using 813A5D5 anti-CD 105 monoclonal antibody as the primary antibody. The experimental procedure was as in example 5, except that the lung adenocarcinoma tissue was changed to "breast cancer tissue".

The prepared mounting piece was observed by using a fluorescence microscope, and as shown in fig. 3, the CD105 protein is expressed by specific cell membranes in breast cancer tissues, and the CD105 signals of 813A5D5 are all significantly stronger than those of the commercial CD105 group. The commercial CD105 group has weak staining positive signals and can have the problem of low detection sensitivity in some tissues with low malignancy degree or early cancer, while the 813A5D5 anti-CD 105 antibody of the invention has the characteristics of good specificity, strong positive signals and the like, so that the antibody can be scored more easily in IHC staining and can be used for detecting and distinguishing cancers more accurately.

Example 7

813A5D5 affinity assay for anti-CD 105 monoclonal antibody was performed as follows:

(1) the polypeptide antigen of CD105 as shown in SEQ ID NO 1-3 was removed from 4 ℃. Return to room temperature. Different polypeptide molarities were prepared and 100. mu.L/well was added to a 96-well plate.

(2) 813A5D5 antibody was diluted to an initial mass concentration of 0.125. mu.g/mL, the antibody molecular weight was 150kD, and the molar concentration was approximately 0.83 nmol/L.

(3) The diluted 813A5D5 anti-CD 105 monoclonal antibody is added to a 96-well enzyme label plate with polypeptide according to 100 mu L/well, a sealing plate membrane is covered, and the incubation is carried out for 1h at the constant temperature of 37 ℃ so that the reaction reaches the equilibrium.

(4) Taking out the enzyme label plate from the incubation chamber at 37 ℃, discarding liquid, washing with purified water for 5 times, and patting to dry.

(5) According to the instructions of the secondary antibody, diluted HRP-labeled goat anti-mouse IgG is added into the ELISA plate at the rate of 100. mu.L/hole, and the reaction is incubated at the constant temperature of 37 ℃ for 1h to reach the equilibrium.

(6) Taking out the enzyme label plate from the incubation chamber at 37 ℃, emptying the liquid, washing for 5 times, and patting dry the water.

(7) TMB developing solution was added to 100. mu.L of each well, and the reaction was carried out at room temperature for 6 minutes.

(8) 50 μ L of 2M H per well₂SO₄The color development was terminated.

(9) And (4) reading the OD value at 450nm on a microplate reader, collating the data and analyzing the result.

The results show that the affinity constant of 813A5D5 anti-CD 105 monoclonal antibody at a polypeptide molar concentration of 0.41. mu. mol/L is: 8X 10^-7(mol/L)。

Example 8

The amino acid sequence and the DNA sequence of the 813A5D5 anti-CD 105 monoclonal antibody disclosed by the invention are shown in SEQ ID NO: 4-7, constructing an expression vector of the CD105 antibody in vitro. The expression vector is amplified and extracted by escherichia coli, endotoxin is removed, Expi-293F cells are transfected, culture is continued after transfection, cell supernatant is collected, and the monoclonal antibody with CD105 resistance specificity is obtained after purification by adopting an affinity chromatography.

The specific method comprises the following steps:

(1) construction of an expression vector: the light chain variable region (VL) sequence (SEQ ID NO: 7) of the anti-CD 105-specific monoclonal antibody was constructed to the kappa chain constant region (SEQ ID NO:8) of the murine antibody light chain, and the heavy chain variable region (VH) sequence (SEQ ID NO: 6) of the anti-CD 105-specific monoclonal antibody was constructed to the constant region of the murine antibody heavy chain, preferably murine IgG1(SEQ ID NO: 9). Adding optimized signal peptide into the N end of the heavy chain/light chain amino acid of the recombinant antibody for secretory expression, performing codon optimization on the amino acid of the recombinant antibody, adding a Kozak sequence GCCGCCACC into the 5' end of the nucleotide, adding EcoRI/HindIII enzyme cutting sites of pcDNA3.4 into the two ends of the nucleotide, and connecting the synthesized gene to a pcDNA3.4 vector after enzyme cutting to obtain an expression vector.

(2) Transfection: the expression vector is amplified and extracted by escherichia coli, endotoxin is removed, and the expression vector is amplified and extracted by the plasmids: Expi-293F cells were transfected at 1 μ g/ml medium. The Transfection reagent used was Expifactamine 293Transfection Kit (Theromfisher, Lot #: A14524) with a cell density of 25 x 10 at Transfection⁵cells/ml, expression enhancers Enhancer1 and Enhancer2 were added 18h after transfection, and cell supernatants were collected 5 days after transfection.

(3) Protein A purification: centrifuging the supernatant at 10000rpm/min at 4 deg.C for 30min to remove cell debris, equilibrating the Protein A column with equilibration solution (0.02MPB, 0.15M NaCl, pH7.0) for 10 column volumes, flowing the supernatant through the column at a rate of 2ml/min, washing the combined column with equilibration solution for 5 column volumes, adding eluent (0.02M PB, 0.15M NaCl, pH3.0) for elution, and dropping the eluent into a collection tube containing neutralization solution (1M Tris, pH 9.0). The protein eluate was collected, concentrated by ultrafiltration using an ultrafiltration tube (Millipore UFC903096)4000G and buffer was replaced with PBS (HyClone SH30256.01), and the eluate was stored at-20 ℃ after detection by SDS-PAGE. Removing endotoxin, filtering for sterilization, and detecting purity by SDS-PAGE electrophoresis.

The monoclonal antibody against CD105 specificity prepared by the above method was added to cell lysate of human placental tissue h.placenta, and Western Blot detection was performed, which enables detection of a significant CD105 protein band, as in the case shown in fig. 1.

In addition to the above preparation method, when the 813A5D5 anti-CD 105 monoclonal antibody of the invention is produced in a large scale, a plasmid vector can be constructed according to the antibody coding sequence disclosed by the invention, the plasmid vector is transferred to cells to obtain a stable expression cell strain, the stable expression cell strain is fermented and cultured in a fermentation tank to obtain cell supernatant or lysate of the stable expression cell strain, and the cell supernatant or lysate is purified by affinity chromatography to produce and prepare the 813A5D5 anti-CD 105 monoclonal antibody.

Finally, it should be noted that: the 813A5D5 anti-CD 105 monoclonal antibody is prepared by any existing method or possible future methods on the basis of the amino acid sequence of the 813A5D5 anti-CD 105 monoclonal antibody disclosed in the application and the DNA sequence encoding the amino acid sequence by the skilled person in the art, and the method is also covered by the protection scope of the patent of the application. Similarly, any form of commercial application of the 813A5D5 anti-CD 105 monoclonal antibody of the present invention, including but not limited to kits, antibody chips, and the like, should be covered by the protection scope of the present application.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

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<400> 5

gacatcgtgc tgacccagag caccgccatc atgagcgcca gccccggcga caaggtgacc 60

atgacctgca gcgccagcag cagcatcagc tacatccact ggtaccagca gaagcccggc 120

accagcccca agagatggat ctacctgacc agcaagctgg ccagcggcgt gcccgccaga 180

ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagcat ggaggccgag 240

gacgccgcca cctactactg cgtgcagaga agcttctacg actggacctt cggcggcggc 300

accaagctgg agatcaag 318

<210> 6

<211> 116

<212> PRT

<213> Artificial Sequence

<400> 6

Glu Val Gln Leu Gln Glu Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr

20 25 30

Trp Met His Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Gly Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Arg Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Val Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Gly Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 7

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 7

Asp Ile Val Leu Thr Gln Ser Thr Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Asp Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Leu Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Val Gln Arg Ser Phe Tyr Asp Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 8

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 9

<211> 324

<212> PRT

<213> Artificial Sequence

<400> 9

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys