Screening Of Phage Displayed Library Using BLyS_134-285 And Studies On Mutants Of BLyS Receptor

B lymphocyte stimulator (BLyS) is a newly identified member of the tumor necrosis factor (TNF) family and plays a key role in stimylating B-cell prolifetation, differentiation and survival. The over-expression of BLyS can lead to a breakdown in tolerance and autoimmunity. In this study, firstly, the bioactive BLyS_134-285 was expressed in E.coli solubly. A phage-displayed peptide library was screened using BLyS_134-285 and a binding peptide was obtained. Then the amino acid sequences of the peptide were compared with B cell maturation antigen (BCMA) sequence, which is the receptor of BLyS. After identifing the research site four mutants of BCMA were constructed by site-directed mutation. The key amino acid positions that have much effects on BLyS_134-285 binding were found. At last a phage display single chain fragment variable library was screened using BLyS_134-285 and FP248 as selective molecules. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. Generally, our work was conducted as follow:(1) BLyS_134-285 was expressed in E.coli BL21 (DE3) solubly at lower inducing temperature. Ni-NTA resin was administed for BLyS_134-285 purification with a final purity, about 80% and the puried BLyS_134-285 was proved to be an effective stimulator...