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Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11
Abstract:
Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.
Insights
Monoclonal antibody 3H11 targets a novel cancer antigen. This antigen is highly expressed in tumors and embryonic tissues but not normal adult tissues, suggesting a role in cancer development and potential for biotherapy.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Monoclonal antibody (MAb) 3H11 specifically binds to various cancer cells.
- MAb 3H11 is clinically used for cancer detection.
- Cloning the target antigen is crucial for understanding tumor biology and developing cancer biotherapies.
Purpose of the Study:
- To identify and characterize the antigen recognized by MAb 3H11.
- To investigate the antigen's expression patterns and potential role in cancer.
Main Methods:
- Screening a human gastric cancer cell line cDNA library using MAb 3H11.
- Expressing a fusion protein of the antigen and analyzing its interaction with MAb 3H11.
- Utilizing Rapid Amplification of cDNA Ends (RACE) and nested PCR to obtain the full-length cDNA.
- Performing Northern blot and RT-PCR to analyze mRNA expression.
- Generating antibodies against the prokaryotically expressed antigen.
Main Results:
- A positive clone expressing a 0.5 kb cDNA fragment recognized by MAb 3H11 was isolated.
- The full-length cDNA (2156 bp) encodes a protein of 589 amino acids with no highly homologous genes in GenBank.
- The antigen's mRNA is widely distributed in embryonic and cancerous tissues but absent in corresponding normal tissues.
- The antigen exhibits low differentiation and high proliferation characteristics.
- Prokaryotic expression of the antigen showed low immunogenicity in mammals.
Conclusions:
- A novel cancer-associated antigen recognized by MAb 3H11 has been identified and cloned.
- The antigen's restricted expression in tumors and embryonic tissues suggests its role as an expression regulator.
- The antigen's characteristics indicate potential as a target for cancer biotherapy.