人可溶性B淋巴细胞刺激因子基因的克隆及在大肠杆菌中的表达

Cloning and expression of soluble B lymphocyte stimulator

目的:钓取人B细胞刺激因子基因,构建其表达的大肠杆菌工程菌,并体外表达、纯化可溶性B细胞刺激因子方法:从人外周血单个核细胞(PBMC)中提取总RNA并逆转录得到cDNA,用PCR扩增目的基因,连接到原核生物表达载体pET-30a上。制备质粒后,酶切、进行菌落PCR及DN测序对其进行鉴定。然后转化大肠杆菌BL21(DE3)并表达对纯化的目的蛋白进行肽质量指纹分析、鉴定,并进一步做体外B细胞刺激试验。结果:RT-PCR钓取出可溶性B细胞刺激因子基因片段。DNA测序结果与报道序列完全一致。在IPTG诱导下,目的基因在工程菌中表达。利用镍金属螯合琼脂糖凝胶亲和层析柱分离。纯化目的蛋白的肽质量指纹图经Mascot搜索与B细胞刺激因子吻合。纯化的蛋白在体外可刺激B细胞增殖。结论:成功地克隆可溶性B细胞刺激因子基因,表达的纯化目的蛋白是具有生物学活性的B细胞刺激因子。

AIM： To clone and express soluble B lymphocyte stimulator （sBLyS）. METHODS. Total RNA was isolated from peripheral blood mononuclear cells, and used to synthesize cDNA by reverse transcription, sBLyS cDNA was amplified by PCR with specific primers and inserted into a prokaryotic expression vector pET-30a. Recombinant plasmid was transformed into E. coli strain BL21 （ DE3 ）. sBLyS was expressed in E. coli, purified in vitro, and analyzed with peptide mass fingerprinting and Daudi cell proliferation assay. RESULTS： sBLyS cDNA was cloned. Peptide mass fingerprinting of purified BLyS matched with that of BLyS proteins. Purified sBLyS could stimulate Daudi cell proliferation in vitro. CONCLUSION： sBLyS with biological activity was successfully expressed and purified.