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Monoclonal antibody to ets domain transcription factor fev protein and its application
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A monoclonal antibody and protein technology, applied in the direction of anti-animal/human immunoglobulin, microorganisms, biochemical equipment and methods, etc., can solve the problem of rare monoclonal antibodies
Active Publication Date: 2018-07-13
GUANGZHOU FIRST PEOPLES HOSPITAL (GUANGZHOU DIGESTIVE DISEASE CENT GUANGZHOU FIRST PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV THE SECOND AFFILIATED HOSPITAL OF SOUTH CHINA UNIV OF TECH)
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[0005] There are polyclonal antibodies against FEV at home and abroad, but its monoclonal antibodies are rare
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Embodiment 1
[0020] Embodiment 1, establishment of hybridoma cell line
[0021] 1. Experimental materials
[0022] 1. Immunogen: In this example, FEV protein is used as the target protein, and a polypeptide sequence is selected from it. The sequence is as follows: Polypeptide A: CPDPVGDGLFKDGKNPS (SEQ ID NO.1). This polypeptide is prepared by chemical synthesis, and the purity requirement is greater than 90%. . This polypeptide is coupled with KLH to prepare an immunogen.
[0023] 2. Culture medium: DMEM medium was purchased from Hyclone Company; HAT, HT selection medium and pristane were purchased from Sigma Company.
[0025] 4. Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company; PEG4000 was purchased from Fluka Company; HRP-goat anti-mouse IgG antibody was purchased from JacksonImmune Company; other reagents were domestic analytic...
Embodiment 2
[0054] Example 2 Preparation of monoclonal antibody against FEV protein
[0055] 1 Antibody preparation
[0056] Adult Balb / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation hybridoma cells were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.
[0057] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Purify with Protein G~Sepharose CL-4B, the PBS damping fluid of upper column liquid is 20mM, and column chromatography eluent is: the glycine damping fluid of pH2.7,20mM, obtains the monoclonal antibody of anti-FEV protein (will be made from polypeptide The monoclonal antibody produced by sequence A is called clone 6D9).
[0058] Identific...
Embodiment 3
[0067] Example 3 Application of purified antibody to detection of FEV protein in tissue
[0068] Western blot detection
[0069] Use clone 6D9 to detect FEV protein in prostate tissue. Take 2 copies of prostatic hyperplasia tissue and 2 copies of prostate cancer tissue, after being treated with protein lysate, centrifuge to get the supernatant for SDS-PAGE electrophoresis, transfer to PVDF membrane by wet transfer method, and use clone 6D9 purified antibody for Western Blot hybridization detection. There are 3 main bands in prostate tissue samples, respectively at 70kDa, 55kDa and 25kDa, and prostate cancer (pca) expresses a higher amount of FEV than hyperplastic tissue (bph). It can be seen that the anti-FEV antibody 6D9 can be used for the detection and expression identification of FEV in clinical samples.
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Abstract
The invention relates to a hybridoma cell line, a monoclonal antibody generated from the hybridoma cell line and an application of the monoclonal antibody, and in particular relates to an ETS structural domain transcription factor FEV protein monoclonal antibody, a hybridoma cell line excreting the antibody and an application of the antibody. The monoclonal antibody is prepared from an immunogen which is obtained by coupling a polypeptide A sequence designed and synthesized by taking FEV protein as target protein with carrier protein; the polypeptide A sequence is shown as SEQ ID NO.1; the carrier protein is KLH; the polypeptide A sequence of the FEV protein can be recognized by the monoclonal antibody; the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO.3. The monoclonal antibody disclosed by the invention can accurately detect the level of FEV protein in a sample, so that the monoclonal antibody is to be widely applied in clinical detection.
Description
technical field [0001] The present invention relates to a hybridoma cell line and the monoclonal antibody produced therefrom and its application, in particular to the monoclonal antibody directed at the ETS domain transcription factor FEV protein, the hybridoma cell line secreting the antibody and the application of the antibody. technical background [0002] Prostate cancer is a common malignant tumor of the male genitourinary system. Worldwide, the incidence of prostate cancer ranks second among all malignant tumors in men. The incidence of prostate cancer in the United States has surpassed that of lung cancer, becoming the first tumor that endangers men's health. According to the estimate of the American Cancer Society, there were about 217,730 new cases of prostate cancer in the United States in 2010, and 32,050 cases will die of this disease. In Europe, approximately 2.6 million new cases of prostate cancer are diagnosed each year. Prostate cancer accounts for 11% of a...
Claims
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Application Information
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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20
Inventor 钟惟德吴群力
Owner GUANGZHOU FIRST PEOPLES HOSPITAL (GUANGZHOU DIGESTIVE DISEASE CENT GUANGZHOU FIRST PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV THE SECOND AFFILIATED HOSPITAL OF SOUTH CHINA UNIV OF TECH)
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