Strassburg C P, Kasai Y, Seng B A, Miniou P, Zaloudik J, Herlyn D, Koprowski H, Linnenbach A J
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Cancer Res. 1992 Feb 15;52(4):815-21.
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.
GA733-2是一种单克隆抗体定义的40 kDa糖蛋白抗原，与多种起源的癌相关。对互补DNA（cDNA）预测的蛋白质序列进行的疏水性分析表明，GA733-2抗原是一种I型膜蛋白。在本研究中，聚合酶链反应被用于一种策略，即省略跨膜和胞质结构域的cDNA序列，从而将细胞外结构域转化为分泌蛋白。全长和截短的cDNA被克隆到杆状病毒转移载体pVL1392中，并通过同源重组引入苜蓿银纹夜蛾核型多角体病毒。全长cDNA杆状病毒重组体指导一种40 kDa糖蛋白的表达，该糖蛋白局限于感染的草地贪夜蛾细胞，而感染截短cDNA杆状病毒重组体的细胞则大量分泌一种31 kDa糖蛋白到培养基中。重组分泌抗原在体外对单克隆抗体表现出免疫反应性，在小鼠体内表现出与天然抗原相似的免疫原性。毫克量癌相关抗原的简便纯化将能够评估其在癌症患者中的免疫原性。
