新型B淋巴细胞阻断剂（TACI）的蛋白质工程

发布时间：2018-07-18 08:40

【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)是一重要的肿瘤坏死因子超家族成员。增值诱导配体(APRIL,又叫TALL-2、TRDL-1及TNFSF-13)与BAFF的蛋白序列具有33%的同源性。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)是APRIL和BAFF共同的受体。本文中我们进行了人TACI胞外区功能域CRD2基因的克隆,基于受体和Fc的晶体结构,通过计算机模拟技术、分子对接方法,构建了人双受体融合基因酵母表达载体-pPIC9K-CRD2-TACI-IgGFc,实现了重组CRD2-TACI-Fc融合蛋白的诱导表达、纯化后进行了生物学活性的鉴定。旨在为进一步研究BLyS/APRIL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/APRIL拮抗剂奠定基础。该项研究取得的主要结果如下： (1)经PCR成功克隆了编码人TACI的胞外段功能域CRD2基因,测序结果与GenBank结果一致。 (2)扩增的TACI胞外区功能域CRD2与TACI-Fc段基因连接后插入真核表达质粒pPIC9K,成功地构建了重组真核表达质粒pPIC9K-CRD2-TACI-IgGFc。 (3)上述质粒转化毕赤酵母GS115。经甲醇诱导,并优化诱导条件,获得了较高水平的表达。SDS-PAGE分析可见,在46kDa处有目的条带。Western Blot结果表明该蛋白与抗TACI抗体显阳性反应。蛋白序列的质谱分析结果表明,该重组蛋白的一级结构与所设计的DNA序列编码的氨基酸序列一致。ProteinA亲和层析柱进行纯化,得到高纯度的融合蛋白CRD2-TACI-Fc。 (4)纯化的融合蛋白进行CCK-8检测,结果表明CRD2-TACI-Fc和TACI-Fc融合蛋白可以阻断APRIL对Raji B细胞系的促增殖作用,并且CRD2-TACI-Fc较TACI-Fc的阻断作用强。本项研究为进一步探讨SLE等自身免疫疾病的发病机制和生物学治疗方法奠定了基础。
[Abstract]:B lymphocyte stimulator (BLyS), also known as tumor necrosis factor family B cell-activating factor belonged to family (BAFF), is an important member of tumor necrosis factor superfamily. Value-added inducible ligands (APRIL, also known as TALL-2TRDL-1 and TNFSF-13) have 33% homology with BAFF protein sequences. Transmembrane protein activator (transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (B cell maturation antigen, BCMA) are the common receptors of APRIL and BAFF. In this paper, we have cloned the CRD2 gene of human TACI extracellular domain. Based on the crystal structure of receptor and FC, we have used computer simulation technique, molecular docking method. The yeast expression vector -pPIC9K-CRD2-TACI-IgGFcwas constructed, and the recombinant CRD2-TACI-Fc fusion protein was induced and expressed. The biological activity of recombinant CRD2-TACI-Fc fusion protein was identified after purification. The aim of this study is to lay a foundation for further study on the interaction between BLyS / APRIL and its receptors and for the development of BLyS / APRIL antagonists for the treatment of autoimmune diseases. The main results of this study are as follows: (1) the extracellular domain CRD2 gene encoding human TACI was cloned successfully by PCR. The results of sequencing were consistent with those of GenBank. (2) the amplified extracellular domain of TACI, CRD2, was ligated with TACI-Fc gene and inserted into eukaryotic expression plasmid pPIC9K. The recombinant eukaryotic expression plasmid pPIC9K-CRD2-TACI-IgGFc. (3) the recombinant plasmid was transformed into Pichia pastoris GS115. After methanol induction and optimization of the induction conditions, a high level of expression. SDS-PAGE analysis showed that the target band. Western Blot at 46kDa showed that the protein reacted positively with anti-TACI antibody. Mass spectrometry analysis of the protein sequence showed that the primary structure of the recombinant protein was consistent with the amino acid sequence encoded by the designed DNA sequence. The protein A affinity chromatography column was used to purify the recombinant protein. The purified fusion protein CRD2-TACI-Fc. (4) was detected by CCK-8. The results showed that CRD2-TACI-Fc and TACI-Fc fusion proteins could block the proliferation of Raji B cell line induced by APRIL, and CRD2-TACI-Fc was stronger than TACI-Fc. This study lays a foundation for further study of pathogenesis and biological therapy of autoimmune diseases such as SLE.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【相似文献】