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JNCI Journal of the National Cancer Institute 1994 86(7):515-526; doi:10.1093/jnci/86.7.515
© 1994 by Oxford University Press
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Journal of the National Cancer Institute, Vol. 86, No. 7, 515-526, April 6, 1994
© 1994 Oxford University Press

Identification of Partial Complementary DNA Clones Encoding a 59-kd Protein With Characteristics of a Unique Oncofetal Antigen

Haroon-Ur Rashid, Adel L. Barsoum, Tin M. Cao, Joseph H. Coggin, Jr.

Department of Microbiology and Immunology, University of South Alabama, College of Medicine Mobile

Correspondence to: Joseph H. Coggin, Jr., Ph.D., Department of Microbiology and Immunology, 170 CSAB, Mobile, AL 36688.

BACKGROUND: Oncofetal antigens (OFAs) are conserved tumor-associated autoantigens or transplantation antigens present on the surface of all major classes of rodent and human tumors and on midgestational fetal cells but not only normal neonatal or adult human and rodent tissues. A syngeneically derived monoclonal antibody, MAb-115, recognizes murine OFAs of 44 and 200 kd in molecular mass.

PURPOSE: Our goal was to clone and characterize the complementary DNAs (cDNAs) that encode these murine OFAs.

METHODS: Rabbit antiserum raised against purified 44-kd OFA glycoprotein was used to screen, a mouse embryc cDNA-lambda phage expression library. Recombinant phage clones positive for the expression of OFAs were detected by immunohistochemical staining, then isolated and plaque purified. The presence of an OFA-encoding sequence in the recombinant phage was confirmed by specific reaction of the expressed protein with MAb-115. Recombinant fusion protein was purified from the extracts of corresponding lysogens. Rabbit antiserum against purified recombinant fusion protein was raised, and the capacity of this antiserum to detect the expression of OFA on rodent tumor and fetal cells was determined by flow cytometry. In addition, immunoreactivity of tumor bearer and hyperimmune murinesera to bacterially expressed recombinant OFA protein was evaluated by enzyme-linked immunosorbent assay. The OFA-expressing insert DNA from plaque-purified lambdaclones was subcloned into phagemid vectors for sequencing analysis.

RESULTS: Antiserum derived against the isolated recombinant mouse embryo polypeptide mimicked MAb-115 in its specific binding to all OFA-positive rodent tumor and fetal cell lines tested and likewise did not show reactivity to normal adult tissues. This antiserum specifically recognized the native 44- and 200-kd OFAs in extracts of murine lymphocytic lymphoma. Furthermore, sera of tumor-bearing mice or mice immunized with purified OFA or intact, irradiated OFA-positive lymphocytic lymphoma cells also reacted with the recombinant fusion protein. The characterization of the isolated clone included nucleotide sequence information followed by analysis of the deduced primary structure of the protein.

CONCLUSIONS: These data suggest that the isolated cDNA clones encode a distinct gene product which is widely expressed on the surface of tumor and fetal cells and represents the first characterized sequence of a true OFA.

IMPLICATIONS: The availability of this cDNA, encoding a protein expressed only on tumor and fetal cells, provides a direct means to assess biological characteristics of malignant tissue which can be assayed by biochemical, histochemical, and molecular methods. (J Natl Cancer Inst 86: 515–526, 1994)


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