人BCMA基因的克隆、表达及抗体制备

【作者】 郑艳；

【导师】 张伟；

【作者基本信息】 北京协和医学院 ， 免疫学， 2009， 硕士

【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。更多还原

【Abstract】 The human BCMA gene is a type III transmembrance protein composed by 185 amino acids. It belongs to the tumor necrosis factor receptor family. BCMA is a receptor for both APRIL (a proliferation-inducing ligand) and BAFF (B cell activating factor belong to the TNF family), which can stimulate B cell proliferation. Now, the functions of BCMA in B cell proliferation, development, as well as in some tumor cell lines and autoimmune diseases needs further investigation.This purpose of this work was to make antibody against BCMA. Then the antibody will be used to detect BCMA espression level on B cell and in serum of patients with autoimmune diseases.In our study, we extracted total RNA from Raji cell line to amplify the cDNA fragment encoding full length of 185 amino acids of BCMA by RT-PCR. The sequence was inserted into pET-28a (+) and pGEX-4T-1 plasmid. PET-28a (+)-BCMA was transformed into E.coli BL21 (DE3). Most of the expression products are inclusion body. PGEX-4T-1-BCMA transformed into E.coli DH5a, the expression products are the soluble protein. Then we produced the mouse anti-hsBCMA polyclonal serum with this purified soluble fusion protein. When we analyzed polyclonal serum with flow cytometry, we found that BCMA on the cell of Raji can not be recognized by the mouse polyclonal serum. We also synthesized a peptide with 12 amino acids from the extracellular domain of BCMA, immunized rabbit with it and got the anti-multiple peptides polyclonal antibody. After Western blot detecting, we found that the polyclonal antibody can recognize the BCMA inclusion body and the multiple peptides.Next, we will optimize the condition of expression, for example using yeast expression and eukaryotic expressing, in order to product the protein of BCMA with Biological activity, then get the McAbs and polyclonal serum against BCMA. These antibodies may offer a good tool for future basic and clinical research.更多还原