CN-121991230-A - Anti-ER recombinant rabbit monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of immunochemistry, and particularly relates to an ER-resistant recombinant rabbit monoclonal antibody and application thereof, wherein the ER-resistant recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-ER recombinant rabbit monoclonal antibody, a preparation method, application of the anti-ER recombinant rabbit monoclonal antibody in an ER protein detection method or device and the like. The ER recombinant rabbit monoclonal antibody cloned by 658G3A2 has the characteristics of good specificity, strong positive signals and the like, is easier to score in IHC staining, and is more accurate for detecting and distinguishing cancers.

Inventors

• XIE BIN
• ZHANG YANNI
• ZHANG DONGXU

Assignees

• 苏州百道医疗科技有限公司
• 中南大学湘雅医院
• 西安交通大学医学院第二附属医院

Dates

Publication Date

20260508

Application Date

20260408

Claims (10)

1. 1. The anti-ER recombinant rabbit monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9.
2. 2. A coding gene for the anti-ER recombinant rabbit monoclonal antibody of claim 1.
3. 3. The coding gene according to claim 2, comprising a DNA sequence as shown in SEQ ID NO. 6 for encoding the heavy chain variable region of the anti-ER recombinant rabbit monoclonal antibody and a DNA sequence as shown in SEQ ID NO. 7 for encoding the light chain variable region of the anti-ER recombinant rabbit monoclonal antibody.
4. 4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.
5. 5. An expression vector or recombinant plasmid comprising the nucleic acid molecule of claim 4.
6. 6. A host cell transformed or transfected with the expression vector or recombinant plasmid of claim 5.
7. 7. The preparation method of the anti-ER recombinant rabbit monoclonal antibody is characterized in that the expression vector or the recombinant plasmid in claim 5 is adopted to transform or transfect the host cell in claim 6, the transformed or transfected cell is cultured, and cell supernatant is collected and purified to obtain the anti-ER recombinant rabbit monoclonal antibody.
8. 8. Use of an anti-ER recombinant rabbit monoclonal antibody according to claim 1, a coding gene according to claim 2 or 3, a nucleic acid molecule according to claim 4, an expression vector or recombinant plasmid according to claim 5, a host cell according to claim 6 for the preparation of an ER detection device.
9. 9. An ER detection kit comprising the anti-ER recombinant rabbit monoclonal antibody of claim 1 and an immunohistochemical detection reagent.
10. 10. The ER detection kit according to claim 9, which comprises the anti-ER recombinant rabbit monoclonal antibody, horseradish peroxidase labeled secondary antibody, ethylenediamine tetraacetic acid repair solution, catalase blocking solution, 3 '-diaminobenzidine concentrate, 3' -diaminobenzidine buffer, hematoxylin and bluing solution according to claim 1.

Description

Anti-ER recombinant rabbit monoclonal antibody and application thereof Technical Field The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-ER recombinant rabbit monoclonal antibody and application thereof, in particular to application in immunohistochemical detection. Background ER nuclear receptor proteins, namely the estrogen receptor (Estrogen Receptor, ER for short), have a molecular weight of about 66kDa and are core molecules mediating the biological effects of estrogens. It activates downstream signal channel by combining with estrogen, regulates and controls various physiological processes of cell proliferation, differentiation, apoptosis and metabolism, and is widely involved in physiological activities of reproductive system development, bone steady state maintenance, cardiovascular function regulation, etc. Meanwhile, ER dysfunction is closely related to the occurrence and development of various diseases, and has important pathological significance in tumors, metabolic diseases and reproductive system diseases. Excessive activation of ER is a key driver of hormone-dependent tumorigenesis development, typically represented by breast cancer and endometrial cancer, and is associated with ovarian cancer, prostate cancer, and the like. However, at present, an Immunohistochemical (IHC) method is often used clinically to carry out in-situ detection on ER protein, and the expression condition of the ER protein in cells is observed through a microscope, so that whether the ER has abnormal expression or not is estimated. However, the anti-ER antibodies used in the prior art have significant drawbacks in terms of sensitivity and specificity, such as low sensitivity due to insufficient affinity of the antibodies. The IHC staining result has the problems of strong subjectivity, poor repeatability and the like in pathological evaluation, and influences the accuracy of disease typing and treatment decision based on ER state. Thus, the invention provides an anti-ER recombinant rabbit monoclonal antibody and application thereof. Disclosure of Invention In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides an anti-ER recombinant rabbit monoclonal antibody which has a wide application range and can accurately recognize ER expression, and an application thereof. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-ER recombinant rabbit monoclonal antibody, a preparation method, application of the anti-ER recombinant rabbit monoclonal antibody in an ER protein detection method or device and the like. In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps: In a first aspect, the invention provides an anti-ER recombinant rabbit monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9. The anti-ER recombinant rabbit monoclonal antibody (ER rabbit source antibody) can be used for immunohistochemical detection, and can identify and detect the expression of ER protein on tumor cells or immune cells with high specificity and high sensitivity. The anti-ER monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-ER recombinant rabbit monoclonal antibody provided by the invention is produced by eukaryotic expression of 293 cells through rabbit hybridoma fusion screening. In the preparation of the anti-ER monoclonal antibody, an antigen for immunizing rabbits (New Zealand white rabbits) is a synthetic polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After the rabbit is immunized, a positive hybridoma cell line capable of secreting monoclonal antibodies with high efficiency is obtained through cell fusion and clone screening, a molecular cloning technology is used for obtaining nucleotide sequences of heavy chain amino acid sequences and light chain amino acid sequences of the antibodies, the nucleotide sequences are constructed on a eukaryotic expression vector, the eukaryotic expression vector is transfected into a 293 cell line through a transfection reagent, cell supernatants are collected, and the cell supernatants are purified through protein A column affinity chromatography, so that the rabbit monoclonal antibodies are obtained. Immunohistochemical detection showed that the antibody specifically recognized ER protein. The anti-ER monoclonal antibody can recognize recombinant ER antigen protein and ER molecules on tumor cells and immune cells, and can be applied to immunohistochemical pathological diagnostic agents. In a secon