Baculovirus recombinant expressing a secreted form of a transmembrane carcinoma-associated antigen.
GA733-2 is a monoclonal antibody-defined, 40-kDa glycoprotein antigen that is associated with carcinomas of various origins. Hydrophobicity analysis of the protein sequence predicted by complementary DNA (cDNA) has suggested that the GA733-2 antigen is a type I membrane protein. In this study, the polymerase chain reaction was used in a strategy to omit cDNA sequences for the transmembrane and cytoplasmic domains, thereby converting the extracellular domain into a secretory protein. Full-length and truncated cDNAs were cloned into the baculovirus transfer vector pVL1392 and introduced into Autographa californica nuclear polyhedrosis virus by homologous recombination. The full-length cDNA baculovirus recombinant directed the expression of a 40-kDa glycoprotein that was confined to infected Spodoptera frugiperda cells, whereas cells infected with the truncated cDNA baculovirus recombinant abundantly secreted a 31-kDa glycoprotein into the culture medium. Recombinant secretory antigen displayed an in vitro immunoreactivity to monoclonal antibody and an in vivo immunogenicity in mice that were similar to native antigen. The facile purification of mg quantities of carcinoma-associated antigen will enable an evaluation of its immunogenicity in cancer patients.
Strassburg
C P
CP
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Kasai
Y
Y
Seng
B A
BA
Miniou
P
P
Zaloudik
J
J
Herlyn
D
D
Koprowski
H
H
Linnenbach
A J
AJ
D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015815:Cell Adhesion Molecules; D002460:Cell Line; D003001:Cloning, Molecular; D000071858:Epithelial Cell Adhesion Molecule; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins