Human Bcma Gene Cloning, Expression And Antibody Preparation

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.