Molecular Mechanism for the Class Switch Recombination of immunoglobulin Gene

我们建立了一株体外培养的B细胞系CH12F3，在转化生长因子-β、白介素4和CD40L的联合作用下，可以诱导类开关重组，培养3d后，50%的细胞从LGM转换为LGA。令人惊讶的是，关节的位置不仅在S地区，而且分布在周边地区。然而，在L地区，我们很少发现这种节理。这些结果表明，该地区仅是S-S复合的起始点。本研究构建了同时含有Sm和Sa序列的重组载体，并将其导入CH12F3细胞。通过对细胞的诱导重组，我们发现SS重组发生在外体DNA中，至少是以较低的频率发生。目前，我们正在重组质粒以提高SS重组的频率。为了分离一个可能控制SS重组事件的新基因，我们发明了新的cDNA消减方法，并鉴定了两个受细胞因子刺激特异性诱导的基因(转化生长因子-β和CD40L)。我们对先天淋巴和生发中心缺陷小鼠(Aly/Aly)进行了生理学分析，发现类开关重组在该小鼠中很少发生。因此，SS的重组需要生发中心B细胞和T细胞的协同调控。

We have developed a cultured B cell line, named CH12F3, which can be induced class switch recombination by the combinational addition of TGF-b, IL-4 and CD40L.As much as 50% of cells switch from lgM to lgA after 3 days of incubation.We have sequenced more than 200 of the DNA fragment which contain the S-S switch joint. Surprisingly the sites of the joint is not only in the S-region but distributed around the peripheral regions. However, we rarely found the joint in l-region. These result suggests that the Sregion works only as the innitiation site of S-S recombination. The place for the whole SS recombination may not be restriced in S sequence.We have constructed a plasmid which contains both Sm and Sa sequence, and introduced it into the CH12F3 cells. By inducing the recombination to the cell, we identified that the SS recombination occurs, at least in low frequency, in the episomal DNA.Presently, we are reconstructing the plasmid to improve the frequency of SS recombination.For isolating a new gene which may control the SS recombination event, we have invented new method of cDNA subtraction, and identified 2 genes which is specifically induced by the stimulation of cytokines (TGF-b and CD40L). Identification of the rolls of these genes are on going.We performed the physiological analysis of the innate lymphnode and germinal center deficient mouse (aly/aly) and found that the class switch recombination rarely occurs' in this mouse. Therefore, for the SS recombination, the cooperative control by B and T cell are required in germinal center.