Molecular cloning and characterization of the gene encoding mouse melanoma antigen by cDNA library transfection.
Molecular cloning and characterization of the gene encoding mouse melanoma antigen by cDNA library transfection.
复制标题
通过 cDNA 文库转染对小鼠黑色素瘤抗原编码基因进行分子克隆和表征。
作者：
H. Hayashi;H. Matsubara;T. Yokota;I. Kuwabara;M. Kanno;H. Koseki;K. Isono;T. Asano;M. Taniguchi
我们已经分离出了一个2.8 kb长的cDNA（H52），编码一个80 kDa的小鼠黑色素瘤抗原，该抗原由一种同基因抗B16黑色素瘤单克隆抗体定义，具有阻断抗黑色素瘤细胞毒性T细胞反应的能力。H52转染子用抗体明亮地染色，并且80-kDa分子从转染子中免疫沉淀。北方印迹分析表明，该转录本在C57 BL/6和DBA/2来源的小鼠黑色素瘤细胞、C1300 A/J神经母细胞瘤、L细胞（C3H）和EL-4 T淋巴瘤（C57 BL/6）中被检测到，在BW 5147（AKR）T淋巴瘤中微弱地被检测到，而在其他肿瘤如S913纤维肉瘤中未检测到（C57BL/10）、NIH3T3、70 Z/3前B淋巴瘤和P3U1浆细胞瘤（BALB/c）。由于在胎儿、新生儿和成人来源的正常C57 BL/6组织中未发现转录本，因此H52表达与转化表型相关。然而，没有观察到组织或细胞类型特异性表达。核苷酸序列分析清楚地表明，H52 cDNA编码全长的env基因和长末端重复区的内源性亲嗜性小鼠白血病前病毒的AKV型，这是在C57 BL/6缺陷。与AKV相比，H52包膜蛋白有几个氨基酸变化，其中之一位于优先与MHC分子相关的env 14肽区域，这表明即使在H52阳性肿瘤中抗体反应性差异的可能原因。我们还证明了针对H52转染子的CTL杀死B16黑色素瘤。因此，上述结果直接证明，即使是内源性自身分子，当组成型表达时，也确实充当肿瘤抗原。
We have isolated a cDNA (H52) of 2.8-kb-long encoding an 80-kDa mouse melanoma Ag that is defined by a syngeneic anti-B16 melanoma mAb with an ability to block anti-melanoma cytotoxic T cell responses. H52 transfectants were brightly stained with the antibody, and the 80-kDa molecule was immunoprecipitated from the transfectants. Northern blot analysis showed that this transcript was detected in mouse melanoma cells of C57BL/6 and DBA/2 origin, C1300 A/J neuroblastoma, L cell (C3H) and EL-4 T lymphoma (C57BL/6), faintly in BW5147 (AKR) T lymphoma, but not in other tumors, such as S913 fibrosarcoma (C57BL/10), NIH3T3, 70 Z/3 pre-B lymphoma, and P3U1 plasmacytoma (BALB/c). Since the transcripts were not found in normal C57BL/6 tissues of fetus, newborn, and adult origin, the H52 expression is associated with transforming phenotypes. However, no tissue- or cell type-specific expression was observed. Nucleotide sequence analysis has clearly demonstrated that H52 cDNA encodes the full length of the env gene and long terminal repeat region of endogenous ecotropic murine leukemia provirus of AKV-type, which is defective in C57BL/6. The H52 envelope protein has several amino acid changes compared to those of AKV, one of which is in the env 14 peptide region preferentially associated with MHC molecule, suggesting the possible reason for the difference of antibody reactivity even in H52-positive tumors. We also demonstrate that CTL against H52 transfectant kills B16 melanoma. Thus, the above results are direct evidence that even the endogenous self molecule, when constitutively expressed, does act as a tumor Ag.