人B淋巴细胞活化相关新基因的克隆

Cloning of Human B Lymphocyte Activation- related Novel Gene

分离新的与 B细胞活化相关基因。方法采用差异显示反转录 PCR（ DDRT－ PCR）技术对人扁桃体活化和静止 B细胞 mRNA的差异表达进行分析，差异显示的片段经过 Northern杂交验证后，作为探针进行人活化 B细胞 cDNA文库的筛选。结果差异显示分析共获得明显的差异表达的标签序列（ expressed sequence tag, EST） 62条，其中主要在静止 B细胞表达的有 32条，在活化 B细胞表达的有 30条。经 Northern杂交验证，共获得阳性的片段 25条。以在活化 B细胞中高表达的 EST30为探针，经 3轮筛选人活化 B细胞文库后获得 1个新的全长为 2 048 bp的 cDNA克隆。该克隆含有 1个 630 bp的开放读码框。其推断的氨基酸序列 N端与酵母的动力蛋白 KAR3部分同源。结论克隆了 1条可能与 B细胞活化相关的新基因。

Objective To clone the novel activation- related gene of B lymphocyte. Methods The differential display reversal transcription PCR (DDRT- PCR) technique was applied to analyse the expression difference of mRNA between resting and activated B lymphocyte from human tonsil. The positive differential display cDNA fragment identified by Northern- blotting was chosen as probe to filtrate human activated B lymphocyte cDNA library. Results Sixty two differential display cDNA fragments(expressed sequence tag, EST)were obtained. Thirty- two of them were mainly expressed in resting B lymphocyts and thirty were expressed in activated cells. Twenty- five were positive ones after identification by Northern blot analysis. A novel cDNA clone was obtained after using EST30 as a probe to filtrate the human activated B cell cDNA library. The whole cDNA clone was 2 048 bp in length and contains a 630 bp open reading frame. The N end of the deduced amino acid sequence was homologous with KAR3 protein which is a member of kinesins superfamily in yeast. Conclusions A novel possible activation- related gene in human B lymphocyte was obtained.