Clarke S H, Kenny J J, Sieckmann D G, Rudikoff S
J Immunol. 1984 Mar;132(3):1544-9.
Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.
表达xid基因的小鼠对磷酸胆碱（PC）偶联的钥孔戚血蓝蛋白（KLH）表现出改变的免疫反应。它们针对PC-KLH的反应中，不到25%是PC特异性的，并且这些抗体中的大多数缺乏正常情况下占主导的T15独特型。这些发现表明，免疫缺陷小鼠在其抗PC反应中可能使用与正常小鼠不同的可变区基因。为了检验这种可能性，我们通过Southern印迹分析对编码PC-VH区的基因家族进行了特征分析，并确定了由SP2/O细胞系与PC-KLH免疫的CBA/N脾细胞融合产生的一种T15、IgG2、PC特异性杂交瘤（1B8E5）的氨基酸序列和结合的精细特异性。使用PC-VH探针（S107 VH）对CBA/N小鼠的DNA进行Southern印迹分析，结果显示其杂交模式与正常CBA/J小鼠的DNA几乎相同，这表明CBA/N小鼠不存在PC-VH基因的大片段缺失。对1B8E5抗体的分析表明，该抗体的结合特异性和相对亲和力与T15、M167-M511和M603家族的抗PC抗体不同。重（H）链可变区的完整氨基酸序列表明，1B8E5使用的VH片段与T15（C3）的等位基因形式相同，但有一个独特的由三个氨基酸组成的D区，并使用JH1连接片段。与正常小鼠的PC特异性抗体相比，DH和JH区都不寻常，正常小鼠的PC特异性抗体有一个由五到八个氨基酸组成的D区，并使用JH1连接片段。1B8E5轻（L）链的氨基末端序列表明，这种抗PC抗体携带Vk3亚组L链。此前尚未发现来自该亚组的链与PC结合抗体相关联。因此，1B8E5中表达的Vk、DH和JH片段使得该杂交瘤在迄今为止研究的抗PC抗体方面具有独特性，并表明在近交系小鼠中存在使用“不寻常”V基因片段的其他PC特异性抗体。
