cloning and expression of a phage display selected single chain antibody for cd19

Introduction: cd19 is an important antigen in manners of immunotherapy and b cell development. studies showed that presence of cd19 is essential for b cell differentiations in various stages of a b lymphocyte. in most b cell associated malignancies, cd19 is expressed in normal to high levels making it a strong marker for targeting malignant b cells. single chain antibodies are a derivative of antibody which only composed of variable regions of the antibody joined to each other by a polypeptide linker. they have been used for various purposes such as diagnostics, therapy and these act like a targeting part in binding to other molecules. production of this binding molecules in e.coli expression systems have been challenging because of inability of these hosts to correctly fold the recombinant protein. therefore, expression and purification condition that improve the solubility of scfvs in this expression system may enable us to obtain a higher yield of functional scfvs for in vitro and in vivo application.materials and methods: in this study, we used a pet expression system induced by iptg in bl21 (de3) strain to express our phage display derived anti-cd19 scfvs. for this purpose, we cloned and expressed three clones of scfvs selected by soluble panning of a human scfv library against our target (cd19 extracellular domain) in phage display. after selection of the positive colonies, bacterial crude extracts of each colony were prepared and their affinity was checked with elisa against cd19.results: pz7 was selected for cloning in pet28a vector, expression in bl21 and purification as it had highest affinity in crude extract elisa. we observed the ~750 bp fragment of scfv after cloning in pet28a vector. appropriate protein size was checked in sds-page before and after purification with ni-nta. a protein band of ~27 kda was confirmed in sds-page and western blotting. furthermore, the sequence analysis showed that scfv pz7 belonged to the human immunoglobulins family which our scfv library has derived from.conclusion: successful implementation of the pet28a vector enabled efficient cloning and expression of a cd19-specific scfv antibody. the hybrid protein purification method employed demonstrates its potential for diverse protein types.