Construction, Expression, Purification And Function Of Single Chain Variable Fragments (ScFv) Against Human CD28 Antigen

The mouse monoclonal antibodies will produce Human Anti-Mouse Antibody, can't get into the target part of the patients bodies because of its bigger size. Recombinant antibody technology has made possible the design of antibodies that can overcome the limitations of diagnostic and therapeutic monoclonal antibodies. Single chain antibodies (ScFvs) are recombinant antigen-binding molecules that retain the VH and VL domains of whole IgG molecules connected by a short polypeptide linker. They have been shown to have reduced immunogenicity, improved penetration into a solid tumor and have a shorter half life in plasma because of its smaller size. In clinical trials, they are ideal for diagnostic and therapeutic applications.In our research, anti-CD28-ScFv-gene and recombine pET32a-anti-CD28-ScFv expression vector have been constructed. SDS-PAGE and Western-blot analysis showed that the recombinant anti-CD28-ScFv gene was expressed in E.coli BL21. ScFv expression was in the form of an inclusion bodies and the purified protein was obtained after a series purification steps including inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding. The purified anti-CD28 ScFv can specifically bind the CD28 that is expressed on the surface of peripheral blood mononuclear cells(PBMC), and can stimulate PBMC to proliferate. That makes a solid fundantion to develop an anti-cancer reagent.