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Mouse anti-human CEA (Carcino-Embryonic Antigen) monoclonal antibody and hybridoma cell strain secreting same

A hybridoma cell line and monoclonal antibody technology, applied in the field of bioengineering, can solve the problem of no early diagnosis value for tumor diseases

Inactive Publication Date: 2013-06-26
TIANJIN SUNGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CEA determination is mainly used for clinical monitoring of colon cancer, rectal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, lung cancer, breast cancer and medullary thyroid cancer, and can also be seen in tumors such as choriocarcinoma, bone cancer, prostate cancer and ovarian cancer CEA, but it has no early diagnostic value for these neoplastic diseases

Method used

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  • Mouse anti-human CEA (Carcino-Embryonic Antigen) monoclonal antibody and hybridoma cell strain secreting same
  • Mouse anti-human CEA (Carcino-Embryonic Antigen) monoclonal antibody and hybridoma cell strain secreting same
  • Mouse anti-human CEA (Carcino-Embryonic Antigen) monoclonal antibody and hybridoma cell strain secreting same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Obtaining of hybridoma cell line 10E1 and the monoclonal antibody it produces

[0023] 1. Antigen preparation

[0024] (1) Obtain the target gene

[0025] In this embodiment, according to the coding frame of the gene sequence of CEA (NM_004363.2), design 1 pair of specific primers:

[0026] Primer 1: 5'-AATGGATCCATGGAGTCTCCCTCGGCCC-3' (SEQ ID NO: 1)

[0027] Primer 2: 5′-ATCCTCGAGCTATATCAGA GCAACC-3′; (SEQ ID NO: 2) Trizol Reagent was used to extract total RNA from human adipose tissue, and the total RNA was reverse-transcribed into cDNA, and the CEA gene was amplified by PCR using cDNA as a template.

[0028] (2) Construction of recombinant expression vector

[0029] The PCR product obtained in step (1) was double digested and recovered, and ligated into the expression vector PET28a under the action of T4 DNA ligase to construct the recombinant plasmid PET28a-CEA.

[0030] (3) Obtain expression strains containing recombinant expression plasmids

[0031]...

Embodiment 2

[0051] Example 2: Application detection of monoclonal antibody immunohistochemistry

[0052] Using CEA monoclonal antibody, pathological sections were made according to conventional methods, and immunohistochemical staining was performed on paraffin sections of human colon adenocarcinoma.

[0053] The specific method is:

[0054] (1) Dewaxing

[0055] The slices were dewaxed in xylene I, II, III in turn for 5 minutes each time, moved to absolute ethanol I, II for 4 minutes each, moved to 95% alcohol for 4 minutes, moved to 85% alcohol for 4 minutes Minutes, then moved to 70% alcohol to soak for 4 minutes, rinsed with running water for 2 minutes.

[0056] (2) Antigen retrieval

[0057] Put the washed tissue slices into a pressure cooker, add about 3000ml of citrate antigen repair solution (pH6.0), heat on high heat and boil, then turn to low heat and keep boiling for 3 minutes, then turn off the switch of the induction cooker for two minutes Finally, move the pressure cooke...

Embodiment 3

[0067] Example 3: Sequencing of variable regions of monoclonal antibodies

[0068] Synthesize the following primers based on the constant region sequence of the antibody gene:

[0069] zh07 5'-GGGGATATCCACCATGGRATGSAGCTGKGTMATSCTCTT-3' (SEQ ID NO: 3)

[0070] zhr11 5'-GACHGATGGGGSTGTYGTGCTAGCTGNRGAGACDGTGA-3' (SEQ ID NO: 4)

[0071] zl01 5'-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3' (SEQ ID NO: 5)

[0072] zlr05 5'-GGATACAGTTGGTGCAGTCGACTTACGTTTKATTTCCARCTT-3' (SEQ ID NO: 6)

[0073] Trizol Reagent reagent extracts 5×10 6 The total RNA of hybridoma cell 10E1 was reverse transcribed into cDNA. Use zh08 and zhr11 as primers to amplify the variable region of the heavy chain of the monoclonal antibody CEA by PCR, and use zl01 and zlr05 as primers to amplify the variable region of the light chain of the monoclonal antibody CEA by PCR. The PCR reactions are all hot-started. The reaction conditions are: 94°C for 5 minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minu...

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Abstract

The invention relates to a mouse anti-human CEA (Carcino-Embryonic Antigen) monoclonal antibody and a hybridoma cell strain secreting the same. The clone number of the hybridoma cell strain secreting the CEA monoclonal antibody is 10E1, and the collection number of the hybridoma cell strain is CGMCC No. 6911. The invention further provides the monoclonal antibody generated by the hybridoma cell strain 10E1. The antibody comprises a heavy-chain variable region and a light-chain variable region, wherein an amino acid sequence of the heavy-chain variable region is SEQ ID NO: 9, and an amino acid sequence of the light-chain variable region is SEQ ID NO: 10. The invention further provides a DNA (Deoxyribonucleic Acid) molecule of SEQ ID NO: 7 and a DNA molecule of SEQ ID NO: 8, wherein the amino acid sequence, namely SEQ ID NO: 9, of the heavy-chain variable region is coded by the DNA molecule of SEQ ID NO: 7, and the amino acid sequence, namely SEQ ID NO: 10, of the light-chain variable region is coded by the DNA molecule of SEQ ID NO: 8. The antibody can be applied to scientific research or the clinical immunohistochemical detection of CEA expression.

Description

technical field [0001] The invention relates to a hybridoma cell line 10E1 secreting a CEA monoclonal antibody obtained by immunizing mice with a prokaryotically expressed CEA protein, and belongs to the technical field of bioengineering. Background technique [0002] CEA (carcino-embryonic antigen) was first discovered from fetal colon cancer tissue, and it is an embryonic carcinogenic antigen, so it is called "carcinoembryonic antigen". The coding gene of CEA is located on chromosome 19, and its expression product is a glycoprotein with a relative molecular mass of 150kDa-300kDa. Studies have shown that the components of CEA are not single, but a protein complex rich in polysaccharides, 45% of which is protein, containing fucose, mannose, galactose and sialic acid. [0003] CEA is synthesized by fetal gastrointestinal epithelium, pancreas and liver cells. The alimentary canal and some tissues in the early stage of the fetus have the ability to synthesize CEA, but the con...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/32C12N15/13
Inventor 张林刚郗日沫尹芝南
Owner TIANJIN SUNGENE BIOTECH
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