阅读说明：本技术 抗ck19蛋白单克隆抗体、细胞系及其制备方法和应用 (anti-CK 19 protein monoclonal antibody, cell line, preparation method and application thereof ) 是由 高惠然 杨清海 陈惠玲 周洪辉 王小亚 于 2020-04-09 设计创作，主要内容包括：本发明涉及生物检测领域,提供了一种抗CK19蛋白单克隆抗体,其重链和轻链可变区的氨基酸序列分别是SEQ ID NO.2和SEQ ID NO.3所示的氨基酸序列。发明人还提供了抗CK19蛋白单克隆抗体的制备方法,选取人CK19氨基酸SEQ ID NO.1序列并与KLH偶联,在其羧基端添加了一个半胱氨酸后,作为免疫原。发明人还提供了一株分泌抗CK19蛋白的杂交瘤细胞系,所述细胞系为小鼠杂交瘤细胞系AbM58058-41A2-PU,保藏号为：CGMCC NO.17489。抗CK19蛋白单克隆抗体,具有高特异性、敏感性,可以特异性识别表达CK19蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。(The invention relates to the field of biological detection, and provides an anti-CK 19 protein monoclonal antibody, wherein the amino acid sequences of heavy chain and light chain variable regions of the anti-CK 19 protein monoclonal antibody are respectively amino acid sequences shown in SEQ ID No.2 and SEQ ID No. 3. the inventor also provides a preparation method of the anti-CK 19 protein monoclonal antibody, selects a human CK19 amino acid SEQ ID No.1 sequence, is coupled with K L H, adds a cysteine at the carboxyl end of the human CK19 amino acid SEQ ID No.1 sequence and serves as an immunogen, and also provides a hybridoma cell line secreting anti-CK 19 protein, wherein the cell line is a mouse hybridoma cell line AbM58058-41A2-PU with the preservation number of CGMCC NO.17489 and the anti-CK 19 protein monoclonal antibody, has high specificity and sensitivity, can specifically identify cells expressing the CK19 protein, and is suitable for immunological detection, particularly immunohistochemical detection.)

1. The anti-CK 19 protein monoclonal antibody is characterized in that the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes CK19 protein.

4. The monoclonal antibody according to claim 3, which specifically recognizes the amino acid sequence shown as SEQ ID No.1 in the CK19 protein.

5. A monoclonal antibody against CK19 protein is prepared from the hybridoma cell line whose preserving number is CGMCC NO. 17489.

6. The monoclonal antibody of claim 1, wherein the anti-CK 19 protein is a mouse IgG2a subtype monoclonal antibody.

7. A preparation method of a monoclonal antibody against CK19 protein is characterized in that an amino acid sequence shown as SEQ ID NO.1 in CK19 protein is selected, cysteine is added to the carboxyl end of the amino acid sequence, and the amino acid sequence is coupled with carrier protein K L H to serve as immunogen.

8. A hybridoma cell line for secreting monoclonal antibodies against CK19 protein is a mouse hybridoma cell line AbM58058-41A2-PU, and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO. 17489.

9. The anti-CK 19 protein monoclonal antibody of any one of claims 1-6, for use in immunodetection of CK19 protein.

10. The immunoassay of claim 9, wherein said immunoassay comprises immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Technical Field

The invention relates to the field of biological detection, in particular to an anti-CK 19 protein monoclonal antibody, a cell line, a preparation method and application thereof.

Background

CK19 has a molecular weight of 40kDa, and is mainly used for labeling various simple epithelia including glandular epithelia and is mainly used for diagnosing adenocarcinoma. The liver cells do not express CK19, so the liver cells can be used for identifying liver cancer and metastatic adenocarcinoma. The CK19 is mainly applied to diagnosis of 1 thyroid follicular papillary carcinoma and follicular carcinoma, 2 cholangiocellular carcinoma and hepatocellular carcinoma, and 3 chordoma and parachulioma.

Disclosure of Invention

The inventor provides an anti-CK 19 protein monoclonal antibody, wherein the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

Further, the monoclonal antibody specifically recognizes the CK19 protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID NO. 1.

Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO. 17489. The cell line is a mouse hybridoma cell line AbM58058-41A2-PU, and is classified and named as: a mouse hybridoma cell line which has been deposited at 3.04.2019 in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and which is prepared by the institute of microbiology, China academy of sciences, No.3, West Lu 1 Hospital, North Kyoho, Beijing, and the township.

Further, the anti-CK 19 protein is a mouse IgG2a subtype monoclonal antibody.

The inventor also provides a preparation method of the anti-CK 19 protein monoclonal antibody, which comprises the steps of selecting an amino acid sequence shown as SEQ ID NO.1 in the CK19 protein, adding a cysteine at the carboxyl terminal of the amino acid sequence, and coupling the amino acid sequence with a carrier protein K L H to serve as an immunogen.

The inventor also provides a hybridoma cell line for secreting anti-CK 19 protein, wherein the cell line is a mouse hybridoma cell line AbM58058-41A2-PU, the cell line is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 17489.

The inventor also provides the application of the anti-CK 19 protein monoclonal antibody in CK19 protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Different from the prior art, the technical scheme selects an amino acid sequence KAA L EDT L AETEARFGAQ L AHIQA (SEQ ID No.1) region which is different from other similar proteins and has proper length and special antigenicity as an antigenic peptide according to the combined structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and a secondary structure of the CK19 protein and DNA, adds a cysteine at the carboxyl terminal of the region, is coupled with K L H, is used as an immunity source CK19-K L H to immunize mice, and obtains a monoclonal cell line AbM58058-41A2-PU for efficiently secreting the anti-CK 19 protein monoclonal antibody through cell fusion, screening and subcloning, and the anti-CK 19 protein monoclonal antibody secreted by the cell line, wherein the antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CK19 protein and is suitable for immunological detection, particularly for immunohistochemical detection.

Drawings

FIG. 1 shows the result of immunoblot detection of antibody secreted by AbM58058-41A2-PU hybridoma.

FIG. 2 is a graph showing the results of immunohistochemical staining of papillary thyroid cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 3 is a graph of immunohistochemical staining results for colon cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 4 shows the immunohistochemical staining results of gastric cancer sample 1 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 5 shows the immunohistochemical staining results of gastric cancer sample 2 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

Detailed Description