Neutralization Antibodies Against The Binding Of B Lymphocyte Stimulator(BlyS) And Its Recepter Panned By Phage Display From An ScFv Antibody Library

BlyS(B lymphocyte Stimulator) is a number of TNF super family, elevated levels of BlyS may be involved in the pathogenesis of B cell-mediated autoimmune diseases. In this study, we aim to identify and characterize a mouse antibody that can inhibit the binding of BlyS and its receptor BCMA by phage display from an scFv antibody library.Total RNA was prepared from the spleen of BlyS immuned mouse by using RNA purification kit. First-strand cDNA was synthesized from total RNA by using cDNA synthesis kit. Then PCRs were set up for synthesizing mouse heavy chain(VH) gene repertoires and light chain(VL) gene repertoires. The scFv genes were generated by assembly of VH, the (Gly4Ser)31inker, and VL fragment though over-lap PCR. The scFv genes were digested with sfiI, and ligated into the phage-display vector pCGMT that had been cut with the same restriction enzyme. The ligated products were electroporated into E.coli XL1-Blue cells to yield 105 clones antibody library.BlyS, at 5 μg/ml in PBS ,was immobilized on immunotube,BlyS-binding phage was isolated by 5 sequential rounds of biopanning,the positive clones increased from 55% of the third round to 100% of the fifth round.Six clones(3-5,4-1,4-5,4-12,4-16,4-19) that have not cross interactions with other proteins were characterized by ELISA.The nucleotide sequences encoding the Vh and VL chains were determined. Analiysis of the 6 different clones demonstrated that the sequences of 4-1,4-5,4-12,4-16,4-19 are almost same,but 3-5 is different from them.This applied the richment of BlyS antibody.The scFv genes of 3-5and 4-16 were subcloned into expression vector pTc and transformed into E.coli BL21(DE3) to express soluble scFv. The inhibition activity of the soluble scFv were analized by ELISA and cytological experiment,the result indicate that 3-5 can inhibit the binding of BlyS and its receptor BCMA.