[慢性B淋巴细胞白血病患者Ig Fab基因的克隆及其可变区序列分析]

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

作者信息

Zhu Huifen, Wang Feng, Zhang Yue, Shen Guanxin

机构信息

Department of Immunology, Tongji Medical College, Huazhong University of Technology and Science, Wuhan 430030, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2002 Sep;23(9):474-9.

PMID:12482359

Abstract

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.

摘要

目的

对慢性B淋巴细胞白血病患者外周血单个核细胞（PBMC）的Ig可变区进行序列分析。

方法

从慢性B淋巴细胞白血病患者的PBMC中提取总RNA，以oligo-dT为引物从RNA合成cDNA。用Taq DNA聚合酶，一组对应Ig FR1的5'特异性引物和对应CH1（Cμ/C）或CL（Cκ/Cλ）的3'引物对cDNA进行扩增，Ig重链（IgH）和轻链（IgL）可变区的PCR产物用ABI PRISM Dye terminator cycle sequencing ready reaction kit和ABI PRISM 310 Genetic Analyzer进行测序。用DNA tools 5.1系统和“国际免疫遗传学数据库”比较IgH和IgL链可变区的基因同源性。

结果

分别从4例和3例患者中扩增出4条轻链和3条重链。用DNA tools系统对4条轻链和3条重链的序列进行同源性分析。轻链序列高度同源。重链序列差异较大。用“国际免疫遗传学数据库”对可变区序列进行同源性分析表明，这些序列与某些B淋巴细胞白血病的独特型基因同源性较高。4个VL基因属于人Ig Vκ亚群I，3个VH基因中的2个属于VH3家族，1个属于VH5家族。