Carcinoembryonic antigen family: expression in a mouse L-cell transfectant and characterization of a partial cDNA in bacteriophage lambda gt11.
Carcinoembryonic antigen family: expression in a mouse L-cell transfectant and characterization of a partial cDNA in bacteriophage lambda gt11.
复制标题
癌胚抗原家族：在小鼠 L 细胞转染子中的表达以及噬菌体 lambda gt11 中部分 cDNA 的表征。
DOI：
10.1073/pnas.84.15.5350
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发表时间：
1987
影响因子：
11.1
通讯作者：
T. Barnett
中科院分区：
文献类型：
--
作者：
M. Kamarck;J. Elting;J. Hart;S. Goebel;P. Rae;M. A. Nothdurft;J. J. Nedwin;T. Barnett
来自腺癌细胞系LoVo的基因组DNA和mRNA用于产生表达癌胚抗原（CEA）的表位的L细胞转染子和噬菌体λ gt 11 cDNA克隆。用荧光激活细胞分选仪（FACS）选择表达CEA的原代和二代L细胞转染子。这些转染子，包括通过多轮FACS分选选择用于高水平CEA表达的一些克隆，表达与所有测试的抗CEA抗体反应的150 kDa的表面蛋白。同时，在λ gt 11中构建LoVo poly（A）+ RNA的cDNA文库，并用抗CEA的多克隆抗血清筛选融合蛋白。一个阳性克隆，λ cLV 7，被鉴定为与转染DNA特异性杂交。cDNA插入片段（cLV 7）的核酸序列包含两个具有广泛内部同源性的区域，在氨基酸水平上具有大于70%的同一性。cLV 7与LoVo细胞的三种mRNA种类和CEA表达转染子的主要mRNA杂交。将cLV 7与限制性内切酶消化的结肠癌细胞、正常人细胞和人-小鼠体细胞杂交体的基因组DNA杂交，发现存在多条杂交带，其中一条存在于转染细胞中。这些CEA相关序列在肿瘤中不重排，通过体细胞杂交分析，被定位于人类19号染色体。
Genomic DNA and mRNA from the adenocarcinoma cell line LoVo were used to generate L-cell transfectants and a bacteriophage lambda gt11 cDNA clone that express epitopes of carcinoembryonic antigen (CEA). Primary and secondary L-cell transfectants expressing CEA were selected with a fluorescence-activated cell sorter (FACS). These transfectants, including some clones that were selected for high-level CEA expression by multiple rounds of FACS sorting, express a surface protein of 150 kDa that reacts with all anti-CEA antibodies tested. In parallel, a cDNA library of LoVo poly(A)+ RNA was constructed in lambda gt11 and fusion proteins were screened with polyclonal antisera against CEA. One positive clone, lambda cLV7, was identified that hybridized specifically to transfectant DNA. The nucleic acid sequence of the cDNA insert (cLV7) contained two regions of extensive internal homology, with greater than 70% identity at the amino acid level. cLV7 hybridized to three mRNA species of LoVo cells and to a predominant mRNA of the CEA-expressing transfectants. Hybridization of cLV7 to restriction endonuclease-digested genomic DNA of colon carcinoma cells, normal human cells, and human-mouse somatic cell hybrids revealed the presence of multiple hybridizing bands, one of which was present in transfectant cells. These CEA-related sequences are not rearranged in tumors and, by somatic cell hybrid analysis, were mapped to human chromosome 19.