bcmafc作为潜在药物的研究word格式论文.docxVIP

摘 要B 细胞刺激因子(B cell activating factor from the TNF family ,BLyS)，是肿瘤坏 死因子家族中的一个新的成员，是在1999年发现的一种重要的免疫调节因子，它 存在两种形式：一种是膜结合形式，另一种是可溶性蛋白的形式并且它能够调节 B细胞的分化和生长。B细胞成熟抗原(B cell maturation antigen,BCMA)是BLyS的 一种受体，它是一种非糖基化的I整合膜蛋白，主要在成熟的B淋巴细胞中表达。 本文中我们主要鉴定了pET30a-BCMA-Fc重组质粒，重新构建一种pET28a-Fc重 组质粒，并对pET30a-BCMA-Fc,pET28a-Fc两种融合蛋白进行了诱导表达，纯化 和生物学活性的鉴定。本文的主要目的是进一步阐述BLyS与其受体的相互关系， 为未来自身免疫疾病和B细胞淋巴瘤的治疗提供一个实验基础。目前为止，我们已经将制备出的Fc片段成功的插入到了pET28a原核表达质粒 中，成功的构建出了pET28a-Fc重组质粒。之后，我们将pET30a-BCMA-Fc和 pET28a-Fc重组质粒转化到大肠杆菌中，分别用IPTG进行诱导，SDS检测， 蛋白A柱进行了纯化，并获得了高纯度的融合蛋白BCMA-Fc和Fc。最后，我们用 酶联免疫吸附试验分析出BCMA-Fc融合蛋白能够特异性的与BLyS结合，并且随 着蛋白浓度的增加而呈现出一定的递增，而Fc不能与BLyS特异性的结合。此外， 我们还验证了拮抗肽BC能够特异性的抑制BCMA-Fc与BLyS的结合作用，并且其 抑制率随着拮抗肽BC浓度的增大而显著增大；而无关肽NRP则不能特异性的抑 制BCMA-Fc与BLyS的结合，只存在少量的非特异性抑制作用。关键词： BLySBCMA 重组质粒 蛋白表达 功能鉴定ABSTRACTBLyS(B cell activating factor from the TNF family),a novel ligand of the tumor necrosis factor family. It exists both as a type II membrane protein and as a soluble protein，which was found in 1999 and regulates B cell proliferation and survival.BCMA(B cell maturation antigen)is a recptor of Blys,which is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes.In the experimental research,we identified pET30a-BCMA-Fc recombinant plasmid mainly and constructed a new recombinant plasmid(usingNdeⅠand XhoⅠ).Then pET30a-BCMA-Fc and pET28a-Fc recombinant plasmidswere induced by IPTG to express two kinds of corresponding fusion proteins.Subsequently,we have purified the fusion proteins using the protein A column. Last,we also analysed the specific binding capability of fusion proteins of pET30a-BCMA-Fc and pET28a-Fc recombinant plasmids with BLyS and two special small peptides.The main objective of study is further elaborating BLyS interactiing with its receptors. Above all, this research may provide a experimental base for a promising treatment for B cell lymphoma and autoimmune diseases.So far, we successfully constructed a new recombinant plas