人B细胞活化相关基因BC-1514全长cDNA的克隆与原核表达

The cloning and prokaryotic expression of a novel gene BC-1514 from activated B lymphocyte

目的 克隆与B细胞活化相关的新基因及其原核表达。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。将所获得的阳性克隆的编码区经PCR扩增后克隆到原核表达载体pGEX 5X 1中 ,重组质粒经酶切、测序鉴定后转化大肠杆菌BL 2 1,以IPTG诱导表达融合蛋白。结果 以在活化B细胞高表达的EST32为探针 ,经 3轮筛选人活化B细胞文库获得一个新的全长为 15 14bp的cDNA克隆 (命名为BC 15 14)。重组的BC 15 14蛋白可在E .coli中以融合蛋白的形式有效表达 ,其表达量约占细菌总蛋白量的 14.3%左右。BC 15 14cDNA的GenBank的登录号为AF30 44 42。结论 获得了 1条新的与B细胞活化相关的cDNA克隆并在E .coliBL 2 1中得到了有效表达。

Objective The cloning and prokaryotic expression of a novel B lymphocyte activation related gene. Methods The differential display RT PCR(DDRT PCR) technique was applied to analyze the expression difference of mRNA from resting and activated human tonsil B lymphocytes. The positive differential display cDNA fragments identified by Northern blotting were used as a probe to find activated human B lymphocyte cDNA library. The whole coding sequence of positive clone was amplified by PCR and cloned into the pGEX 5X 1 vector. The recombinant plasmid was transformed into E.coli BL 21 and the expression of the fusion protein was induced by IPTG. Results We obtained a novel cDNA clone using EST32, which was mainly expressed in activated B lymphocyte, as a probe to identify the human B cells cDNA library. The positive cDNA clone (named BC 1514) is 1 514 bp in length and contains a open reading frame of 393bp. The fusion protein of BC 1514 was efficiently expressed in E.coli , took about 14.3% of the total bacterial product. The accession number of cDNA 1514 in GenBank is AF304442. Conclusion We obtained a novel activation related gene in human B lymphocyte, of which the coding sequence was efficiently expressed in E.coli BL 21. [