人BCMA基因的克
发布时间：2018-12-13 02:05
【摘要】：BCMA (B cell maturation antigen)是由185个氨基酸残基组成的III型跨膜蛋白。它属于TNF受体家族成员,与其配体B细胞激活因子BAFF (B cell activating factor belonging to the TNF family)或增殖诱导配体APRIL (a proliferation-inducing ligand)结合可刺激B细胞增生。BCMA在B细胞的增殖、发育以及在一些B细胞系肿瘤和自身免疫性疾病中的作用有待进一步研究。 本项目拟制备BCMA抗体,检测BCMA在自身免疫性疾病患者B细胞表面和血清中的表达是否异常,探讨BCMA与自身免疫性疾病的相关性。 通过RT-PCR方法从Raji细胞的总RNA中扩增BCMA的全长cDNA,克隆入原核表达载体pET-28a(+)、pGEX-4T-1。pET-28a(+)-BCMA在大肠杆菌BL21(DE3)中表达,主要以包涵体形式存在于沉淀中。pGEX-4T-1-BCMA在大肠杆菌DH5a中表达为可溶性蛋白,但是用该可溶性BCMA蛋白免疫小鼠6周后,采血进行流式细胞检测发现免疫血清不能识别Raji细胞表面的BCMA蛋白。我们又从BCMA胞外区氨基酸序列中选取12个氨基酸,合成多肽BCMA-12aa,免疫新西兰大白兔,得到了多克隆抗体,Western blot检测发现所得多克隆抗体能够识别多肽BCMA-12aa、可溶性和包涵体形式的BCMA,但是仍不能识别Raji细胞表面的BCMA蛋白。 下一步,建议采用酵母或者真核条件表达有生物活性的BCMA,制备该蛋白的单、多克隆抗体,为自身免疫性疾病和B细胞系肿瘤的基础与临床研究提供有力的工具。
[Abstract]:BCMA (B cell maturation antigen) is a III type transmembrane protein composed of 185 amino acid residues. It belongs to the TNF receptor family and binds to its ligand B-cell activator BAFF (B cell activating factor belonging to the TNF family) or proliferation-inducing ligand (APRIL (a proliferation-inducing ligand) to stimulate the proliferation of B cells. BCMA proliferates in B cells. Development and role in some B cell line tumors and autoimmune diseases need further study. The aim of this study was to prepare BCMA antibody to detect the abnormal expression of BCMA on B cell surface and serum of patients with autoimmune diseases and to explore the correlation between BCMA and autoimmune diseases. The full-length cDNA, of BCMA was amplified from the total RNA of Raji cells by RT-PCR and cloned into the prokaryotic expression vector pET-28a (), pGEX-4T-1.pET-28a () -BCMA, which was expressed in E. coli BL21 (DE3). PGEX-4T-1-BCMA was expressed as soluble protein in Escherichia coli DH5a, but the soluble BCMA protein was immunized with the soluble BCMA protein for 6 weeks. Flow cytometry analysis showed that the immune serum could not recognize the BCMA protein on the surface of Raji cells. We also selected 12 amino acids from the amino acid sequence of extracellular domain of BCMA and immunized New Zealand white rabbits with polypeptide BCMA-12aa,. The polyclonal antibody, Western blot detection showed that the polyclonal antibody could recognize polypeptide BCMA-12aa,. Soluble and inclusion body forms of BCMA, but still unable to recognize the BCMA protein on the surface of Raji cells. The next step is to use yeast or eukaryotic condition to express bioactive BCMA, to prepare mono-polyclonal antibody of the protein, which provides a powerful tool for the basic and clinical study of autoimmune diseases and B-cell line tumors.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1
本文编号：2375676
[Abstract]:BCMA (B cell maturation antigen) is a III type transmembrane protein composed of 185 amino acid residues. It belongs to the TNF receptor family and binds to its ligand B-cell activator BAFF (B cell activating factor belonging to the TNF family) or proliferation-inducing ligand (APRIL (a proliferation-inducing ligand) to stimulate the proliferation of B cells. BCMA proliferates in B cells. Development and role in some B cell line tumors and autoimmune diseases need further study. The aim of this study was to prepare BCMA antibody to detect the abnormal expression of BCMA on B cell surface and serum of patients with autoimmune diseases and to explore the correlation between BCMA and autoimmune diseases. The full-length cDNA, of BCMA was amplified from the total RNA of Raji cells by RT-PCR and cloned into the prokaryotic expression vector pET-28a (), pGEX-4T-1.pET-28a () -BCMA, which was expressed in E. coli BL21 (DE3). PGEX-4T-1-BCMA was expressed as soluble protein in Escherichia coli DH5a, but the soluble BCMA protein was immunized with the soluble BCMA protein for 6 weeks. Flow cytometry analysis showed that the immune serum could not recognize the BCMA protein on the surface of Raji cells. We also selected 12 amino acids from the amino acid sequence of extracellular domain of BCMA and immunized New Zealand white rabbits with polypeptide BCMA-12aa,. The polyclonal antibody, Western blot detection showed that the polyclonal antibody could recognize polypeptide BCMA-12aa,. Soluble and inclusion body forms of BCMA, but still unable to recognize the BCMA protein on the surface of Raji cells. The next step is to use yeast or eukaryotic condition to express bioactive BCMA, to prepare mono-polyclonal antibody of the protein, which provides a powerful tool for the basic and clinical study of autoimmune diseases and B-cell line tumors.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1
【共引文献】
相关期刊论文 前2条
1 郑艳;沈濂;;人B细胞成熟抗原的克隆、表达及纯化[J];癌症进展;2010年06期
2 陈红波,涂胜豪,胡永红;单核苷酸多态性在类风湿关节炎中的研究进展[J];中华风湿病学杂志;2004年03期
相关硕士学位论文 前3条
1 梁伊乐;BAFF及受体BAFF-R在Ⅰ型自身免疫性肝炎中的表达及其临床意义[D];郑州大学;2011年
2 白彩琴;基于CIA大鼠两种抗原短肽特异性自身免疫反应的实验研究[D];南京师范大学;2011年
3 房国祥;环瓜氨酸肽模拟表位自身免疫反应的测定及在RA中的意义[D];江苏大学;2009年
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