Experimental Study Of The Construction, Expression, Purification And Biological Activity Of Anti-PSMA×Anti-FITC Bispecific Diabody

Objective：1. Constructed the gene sequences of the bispecific diabody BsDb(anti-PSMA×anti-FITC) and prokaryotic expression vector pET-28(a)-BsDb; thentransformed it into escherichia coil for expression induced by IPTG and purification.2. To cultivate prostate cancer cell lines and to determine the expression of PSMA; andidentification of the activity of a bispecific diabody. Methods：1. Obtained the heavy chain and light chain gene sequences of anti-PSMA ScFv(ScFv-P)and anti-FITC ScFv(ScFv-F) through analysis of their gene sequences which haveobtained by our team. In Vector NTI9software the ScFv-P heavy chain (PVH)connected by a linker to the ScFv-F light chain (FVL) to build PVH-Linker-FVL，theScFv-F heavy chain (FVH) connected by a linker to ScFv-P light chain (PVL) to buildPVL-linker-FVH. The hybrid ScFv PVH-Linker-FVLand PVL-linker-FVHare connectedto form the gene fragment of BsDb. Design two restriction enzyme cutting sites: aXbaⅠsite in its C-terminal and a XhoⅠsite in N-terminal. The BsDb gene fragmentwas digested with Xba I and Xho I, the plasmid vector pET-28(a) was digested withthe same restriction endonucleases, then formed the PET-28(a)-BsDb and transformedit into E.coli BL21for expression induced by IPTG, to be purified by Ni-NTA spincolumns.2. Human prostate cancer cell lines LNCaP, PC-3and human pancreatic cancer cell linesPANC-1were cultured. The expression of PSMA of prostate cancer cell lines wasdetermined by using western blot.3. The specific binding capability for BsDb to prostate cancer cells was detected by usingimmunohistochemistry and western blot. The specific binding capability for BsDb toFITC was detected by using ELISA.Results：1. Successfully constructed the gene sequences of BsDb and transformed it into E.coliBL21for expression. Finally we worked out the induced conditions which make BsDbto soluble expression. The concentration of IPTG is0.50‰, the other conditions are16℃,150rpm/min12hours. The BsDb which have his-tag was purified by usingcell-free extraction, heat treatment and Ni Sepharose chromatography. The finalconcentrations of imidazole are10,40,120,500mmol/L. The purified BsDb wasgained in500mmol/L imidazole. The concentration of BsDb is181micrograms/mlmeasured by method of BCA. 2. The human prostate cancer cell lines LNCaP, PC-3and the human pancreatic cancercells lines PANC-1are adherent monolayer growth; the results of western blotconfirmed the high expression of PSMA on the plasma membrane of LNCaP cells,while no positive findings on the PC-3cells and PANC-1cells.3. The results of immunocytochemistry and western blot proved that BsDb can specificbind with LNCaP cells on which the PSMA is positive. The results of ELISA provedthat BsDb can specific bind with FITC.Conclusion：1. PSMA is highly expressed on the plasma membrane of the human prostate cancer celllines LNCaP.2. The bispecific diabody BsDb (anti-PSMA×anti-FITC) which can be expressed in theE.coli BL21was successfully constructed by genetic engineering techniques; It hastwo specific binding targets: specific binding to PSMA-positive cells and specificbinding to FITC. It will be useful for vivo tests in the next step.