Analysis of Tumor Rejection Antigen of Murine Myeloma MOPC70A Recognized by Specific Cytotoxic T cell.

鼠骨髓瘤 MOPC70A 特异性细胞毒性 T 细胞 (CTL) 克隆 E10 是从荷瘤 BALB/c 小鼠的脾细胞建立的。 2.将表达cDNA文库从MOPC70A的mRNA克隆到哺乳动物表达载体pMET7中，并分成100个细菌菌落的库。并测试了总共56,000个cDNA克隆在转染COS7转染的H-2基因后通过E10 CTL刺激TNFα产生的能力。 3.克隆了刺激E10产生TNFα和IFNγ的克隆(2D2)。 4. 2D2的序列长度为1398bp。序列同源性分析表明2D2是小鼠凋亡诱导因子(mAIF)。 5.为了鉴定2D2基因编码的抗原CTL表位，研究了2D2基因缺失突变体刺激E10产生TNFα的能力。结果表明，该抗原表位位于2D2的737-870bp区域。通过对737-870bp区域上的合成候选肽的分析，发现九聚物肽DLGPDVGYEA致敏的E10 CTL作为表位。

Murine myeloma MOPC70A specific cytotoxic T cell (CTL) clone E10 was established from spleen cells of tumor bearing BALB/c mouse. 2. Expression cDNA library was cloned from mRNA of MOPC70A into mammalian expression vector pMET7 and divided into pools of 100 bacterial colonies. And the total 56,000 cDNA clones were tested for their ability to stimulate TNFα production by E10 CTL after transfection into COS7 transfected H-2 gene. 3. A clone (2D2) which stimulate E10 to produce TNFα and IFNγ was cloned. 4. The sequence of 2D2 provided 1398 bp long. Sequence homology analysis revealed that 2D2 is murine apoptosis inducing factor (mAIF). 5. To identify antigenic CTL epitope encoded by the 2D2 gene, the ability of deletion mutant of 2D2 gene to stimulate E10 to produce TNFα were investigated. The results suggested that the antigenic epitope resided within the region of 737-870bp of 2D2. From analysis of the synthetic candidate peptide on the region of 737-870 bp, a nonamer peptide DLGPDVGYEA sensitized E10 CTL was found as an epitope.