Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11.
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.
Chen
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Beijing Institute for Cancer Research, Beijing 100034, People's Republic of China.
Shou
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C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D013274:Stomach Neoplasms / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D011994:Recombinant Proteins