Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

Abstract

Background: Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.

Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9

“…The production of soluble scFv-M6-1B9 from E. coli HB2151 was accomplished previously (Tragoolpua et al, 2008). However, the biological functions of soluble scFv-M6-1B9 have yet to be investigated.…”

“…Soluble scFv-M6-1B9 was produced by expressing pComb3X-scFv-M6-1B9 phagemid in the non-suppressor E. coli strain HB2151 as previously reported (Tragoolpua et al, 2008). Briefly, transformed bacteria were grown in SB broth containing ampicillin at 37 1C for 18 h. The precultured bacteria were then transferred and cultivated at the same temperature and medium until the OD 600 reached 1.5.…”

“…Adenovirus expression of scFv-M6-1B9 carrying GFP gene was generated, propagated and purified as described elsewhere (Carlson et al, 2002;Tragoolpua et al, 2008). Titers were determined by plaque titration in 293 cells (Carlson et al, 2002) and the contamination level of wild-type (wt) virus was examined by real-time PCR (Bernt et al, 2002).…”

“…Proteins in the supernatant were precipitated with saturated (NH 4 ) 2 SO 4 in an ice bath and concentrated with Amicon Ultra centrifugal filter units (Millipore, Cork, Ireland). Finally, the antigen-binding affinity of soluble scFv-M6-1B9 was evaluated by ELISA, Western immunoblotting and flow cytometric analysis as described previously (Tragoolpua et al, 2008).…”

“…Recently, we successfully produced soluble scFv against CD147 (scFv-M6-1B9) from its parental mAb, M6-1B9, using bacteria Escherichia coli. We have been able to generate this antibody fragment inside 293A cells to knockdown CD147 cell surface expression (Tragoolpua et al, 2008). However, the biological properties and function of scFv-M6-1B9 have not been fully investigated.…”