Naghibalhossaini Fakhraddin, Pakdel Abbas, Ghaderi Abbas Ali, Saberi Firoozi Mehdi
Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran.
Pathol Oncol Res. 2005;11(4):211-7. doi: 10.1007/BF02893853. Epub 2005 Dec 31.
Carcinoembryonic antigen (CEA), the most widely used human tumor marker, is a heavily glycosylated protein over-expressed by a wide range of tumors. It has been indicated that CEA might be a useful target for human anti-tumor immunotherapy. CEA assay for research as well as clinical trials demands a continuous source of CEA protein preparations. In a multi-purpose research program to provide a reliable source for large production of CEA, we converted the membrane-bound carcinoembryonic antigen into a secretory protein by site-specific mutagenesis. We made the secretory CEA protein by introducing a new stop codon at 99 bp upstream of the original stop codon in CEA cDNA by PCR-based mutagenesis. The glycosylation of recombinant CEA proteins, especially those destined for administration to human trials is crucially important. To produce CEA with the same glycosylation pattern and immunogenicity as the native CEA expressed by human tumors in vivo, the truncated CEA cDNA which does not encode the last C-terminal 33-amino acid hydrophobic domain was transfected into HT29, a human colon carcinoma cell line by the calcium phosphate method. Stable transfectants were selected and pooled. CEA secretion from the cells was verified by analysis of the transfectant culture supernatant for CEA protein. As determined by ELISA, 16 microg/L of recombinant CEA was secreted per 106 transfectants within 48 hrs, an increase over 40 times relative to the untransfected cells. The size of the recombinant CEA secreted by HT29 transfectants in our experiment is identical to that of reference CEA secreted from tumors and is fully antigenic. It seems that the C-terminal truncation does not affect CEA glycosylation in HT29 cells. It is predicted that human cancer immunotherapy using recombinant CEA expressed in this system would be more effective than the commercial protein which is usually prepared from bacterial or other heterologous expression systems.
癌胚抗原（CEA）是应用最广泛的人类肿瘤标志物，是一种在多种肿瘤中过度表达的高度糖基化蛋白。已有研究表明，CEA可能是人类抗肿瘤免疫治疗的一个有用靶点。用于研究及临床试验的CEA检测需要持续供应CEA蛋白制剂。在一项旨在为大量生产CEA提供可靠来源的多用途研究项目中，我们通过位点特异性诱变将膜结合型癌胚抗原转化为分泌型蛋白。我们通过基于PCR的诱变在CEA cDNA原始终止密码子上游99 bp处引入一个新的终止密码子，从而制备出分泌型CEA蛋白。重组CEA蛋白的糖基化，尤其是那些用于人体试验的蛋白的糖基化至关重要。为了产生与人类肿瘤在体内表达的天然CEA具有相同糖基化模式和免疫原性的CEA，将不编码最后33个C末端氨基酸疏水结构域的截短CEA cDNA通过磷酸钙法转染到人结肠癌细胞系HT29中。筛选并汇集稳定转染子。通过分析转染子培养上清液中的CEA蛋白来验证细胞分泌CEA的情况。通过ELISA测定，每106个转染子在48小时内可分泌16 μg/L的重组CEA，相对于未转染细胞增加了40多倍。在我们的实验中，HT29转染子分泌的重组CEA的大小与肿瘤分泌的参考CEA相同，且具有完全抗原性。似乎C末端截短并不影响HT29细胞中CEA的糖基化。据预测，使用该系统表达的重组CEA进行人类癌症免疫治疗将比通常由细菌或其他异源表达系统制备的商业蛋白更有效。
