發(fā)布時間：2018-12-13 02:05

【摘要】：BCMA (B cell maturation antigen)是由185個氨基酸殘基組成的III型跨膜蛋白。它屬于TNF受體家族成員,與其配體B細胞激活因子BAFF (B cell activating factor belonging to the TNF family)或增殖誘導配體APRIL (a proliferation-inducing ligand)結合可刺激B細胞增生。BCMA在B細胞的增殖、發(fā)育以及在一些B細胞系腫瘤和自身免疫性疾病中的作用有待進一步研究。 本項目擬制備BCMA抗體,檢測BCMA在自身免疫性疾病患者B細胞表面和血清中的表達是否異常,探討B(tài)CMA與自身免疫性疾病的相關性。 通過RT-PCR方法從Raji細胞的總RNA中擴增BCMA的全長cDNA,克隆入原核表達載體pET-28a(+)、pGEX-4T-1。pET-28a(+)-BCMA在大腸桿菌BL21(DE3)中表達,主要以包涵體形式存在于沉淀中。pGEX-4T-1-BCMA在大腸桿菌DH5a中表達為可溶性蛋白,但是用該可溶性BCMA蛋白免疫小鼠6周后,采血進行流式細胞檢測發(fā)現免疫血清不能識別Raji細胞表面的BCMA蛋白。我們又從BCMA胞外區(qū)氨基酸序列中選取12個氨基酸,合成多肽BCMA-12aa,免疫新西蘭大白兔,得到了多克隆抗體,Western blot檢測發(fā)現所得多克隆抗體能夠識別多肽BCMA-12aa、可溶性和包涵體形式的BCMA,但是仍不能識別Raji細胞表面的BCMA蛋白。 下一步,建議采用酵母或者真核條件表達有生物活性的BCMA,制備該蛋白的單、多克隆抗體,為自身免疫性疾病和B細胞系腫瘤的基礎與臨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.
【學位授予單位】：北京協和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1

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相關碩士學位論文 前3條

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本文編號：2375676