慢性B淋巴细胞白血病患者免疫球蛋白Fab段基因克隆及其可变区序列分析

目的：分析慢性B淋巴细胞白血病患者外周血单个核细胞免疫球蛋白Fab段基因。方法：提取慢性B淋巴细胞白血病患者外周血单个核细胞RNA,选用与Ig FR1 5′和CH1Cμ3′或CL区（Cκ／Cλ）3′互补的多对引物,通过RT－PCR扩增Ig Fab段基因,进行克隆和序列测定,并通过DNAtools和计算机网络对其可变区基因同源性进行分析和基因家族归类。结果：7例患者中4例外周血单个核细胞扩增出轻链基因,3例扩增出重链基因。所扩增的4条轻链基因均属于κ轻链,3条重链均为μ链基因。利用DNAtools分析软件,对轻链和重链分别进行同源性比较,3条重 链可变区基因差异较大,轻链可变区基因具有一定的同源性。对所扩增的Ig基因进行归类,结果4例患者的轻链均属于VκⅠ亚组;3例患者的重链中2例属VH3家族,1例为VH5家族。结论：慢性Ｂ淋巴细胞白血病患者存在独特的决定簇和相似决定簇。

Cloning of Ig Fab genes of patients with chronic B lymphocytic leukemia and sequences analysis of their variable regions

OBJECTIVE: Sequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia. METHODS: Total RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database". RESULTS: Four light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family. CONCLUSION: Ig genes have idiotype and same disease may have same idiotype.