We try to develop a method for delivering antibody from blood circulation through blood brain barrier to brain. In order to achieve this goal, antibody has to cross cellular membrane of brain capillary endothelial cells twice. As a first step of our study, we examined the ability for scFv antibody to cross cellular membrane of RBL-2H3 cells once and be delivered into the inside of the cultured cells with the help of TAT peptide. TAT peptide was originally found in Tat protein from the HIV-1 virus and known as one of protein transduction domains. First, oligonucleotide encoding TAT peptide was linked to 5' terminal of gene fragment of scFv antibody by PCR technology. TAT-linked scFv gene fragment was subcloned into pET-23b vector and successfully expressed in E. coli as inclusion body. After solubilization and purification, TAT-linked scFv recombinant protein was added to the culture of RBL-2H3 cells. TAT-linked scFv delivered into RBL-2H3 cells was detected by means of immunocytochemistry using fluorescence microscopy. TAT-linked scFv crossed cellular membrane more efficiently than scFv without TAT peptide.
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