【作者】 郭翠霞；

【导师】 孙剑；

【作者基本信息】 天津大学 ， 药物化学， 2008， 硕士

【摘要】 B淋巴细胞刺激因子(B lymphocyte stimulator, BLyS),又称肿瘤坏死因子家族的B细胞活化分子(B cell-activating factor belonged to TNF family, BAFF)是一种重要的肿瘤坏死因子超家族成员。穿膜蛋白活化物(transmembrane activator and CAML interactor, TACI)和B细胞成熟抗原(B cell maturation antigen, BCMA)与BAFF-R (BAFF Receptor)一起成为BLyS的三个受体。本文中我们进行了人TACI全长及胞外区基因的钓取,与人IgG1的Fc段的连接,构建其原核表达载体。并进行了TACI-Fc和BCMA-Fc重组蛋白的诱导表达、纯化及生物学活性的鉴定。旨在为进一步研究BLyS与其受体的相互作用模式和开发用于治疗自身免疫疾病的BLyS拮抗剂奠定基础。该项研究取得的主要结果如下:(1)经RT-PCR法成功克隆了编码人TACI的全长基因,大小约为900bp,测序结果与GenBank结果一致。(2)扩增的TACI胞外区与人IgG1的Fc段基因连接后插入原核表达质粒pET28a,成功地构建了重组原核表达质粒pET28a-TACI-Fc。(3)上述质粒和实验室已构建好的pET28a-BCMA-Fc重组质粒转化大肠杆菌BL21 (DE3),经IPTG诱导,并优化诱导条件,获得了较高水平的表达。SDS-PAGE分析可见,分别在大约40kDa和35kDa处有目的条带。Western Blotting和ELISA实验也显阳性。Protein A亲和层析柱进行纯化,得到高纯度的融合蛋白TACI-Fc和BCMA-Fc。(4)已纯化的融合蛋白送北京进行MTT检测,结果表明TACI-Fc和BCMA-Fc融合蛋白可以阻断BLyS对Nalm-6细胞系的增殖作用,并且TACI-Fc较BCMA-Fc的阻断作用强。更多还原

【Abstract】 B lymphocyte stimulator (BLyS), also known as B cell-activating factor belonged to TNF family (BAFF), is a member of tumor necrosis factor (TNF) family. Transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA), which along with BAFF-R, are the three receptors of BLyS. In this project, we cloned the full length of TACI and ligated the extracellular fragment soluble TACI (sTACI) with human IgG1 Fc fragment, and constructed the prokaryotic expression vectors. Then pET28a-TACI-Fc and pET28a-BCMA-Fc recombinant plasmids were induced by IPTG to express the corresponding proteins. Next, the proteins were purified by Protein A chromatography, and their bioactivity was identified by MTT. We provide experimental base for further study on the action mode of TACI/BCMA with BLyS.In summary, the results of this study are as follows:(1)Clone the full length of TACI. The 900bp cDNA was amplified by RT-PCR, which was consistent with the sequence reported in GenBank.(2)The extracellular fragment soluble TACI (sTACI) was ligated with human IgG1 Fc fragment. sTACI-Fc was subcloned into expression vector pET28a. The prokaryotic expression plasmid pET28a-TACI-Fc was successfully constructed.(3)Recombinant plasmids pET28a-TACI-Fc and pET28a-BCMA-Fc constructed previously were transformed into E.coli BL21 (DE3) respectively. The expression was induced by IPTG and optimal conditions of the induction were achieved. SDS-PAGE analysis showed that about 40kDa and 35kDa fusion proteins were highly expressed. The expressed proteins were identified by Western Blotting and ELISA. The expressed proteins were purified by Protein A chromatography. SDS-PAGE and Western Blotting analysis showed that the target proteins were highly purified.(4)The bioactivity of the purified proteins was indentified by MTT. The fusion proteins could inhibit the proliferation of BLyS to Nalm-6 cell lines. And the inhibitory effect of TACI-Fc fusion protein was more potent than that of BCMA-Fc.更多还原