Selection, Expression, Purification And Function Study Of The ScFv Binding To Prostatic Carcinoma

Abstract In this study, phage display was used to construct a scFv library in order to select the antibodies binding to prostatic carcinoma cells. This work supports the effort to additionally investigate early diagnosis and low-damage therapy of prostatic carcinoma.The recombinant phage-antibody system was used to construct a single-chain Fv fragment (scFv) cDNA library from the total RNA of the mice immunized with purified membrane protein mixture of prostate carcinoma cells PC3, DU145, C4-2. By phage displaying and panning, a phage-scFv named P-9 binding PC-3 cells was selected from the library. The titer difference of scFv P-9 between PC3 and control cells LNCaP was more than 150 times. By sequencing and comparing with the library of mice antibody variable region sequence, we identified that its V_H belonged to the III(c) subset of mice antibody heavy chain variable region gene and V_L belonged to the II subset of mice antibody light chain variable region gene. No other known scFv had the same sequence.By constructing the expression plasmid pTIG-Trx-scFv P-9, scFv P-9 could be expressed as soluble and functional form in cytoplasm of E.coli BL21(DE3). The cell ELISA results detected by anti-His-tag antibody displayed that the scFv P-9 could effectively bind to PC3 cells.The bioactivity of scFv to prostatic carcinoma cells was identified by the MTT cell survival assay. The results were as following: scFv P-9 could inhibit the proliferation of PC3, HeLa, PLA801D cells, but did not affect the growth of LNCaP and normal hepatocyte HL02 cells, and this kind of effect appeared the highest activity at 24 hours after incubation with the cells.To investigate the role of the tumor suppression of scFv P-9, we used an animal model of human cervical carcinoma in BALB/c nude mice. Results showed that the growth of human tumor xenografts in nude mice was dramatically suppressed after treatment with scFv P-9, especially s.c. There was a significant difference in tumor weight between the PBS group and the scFv P-9 group during the observation period. By the histological assay, many tissue necrosis were observed in the tumor tissues of the scFv group, and this could not be found in the PBS group and the control scFv group. It demonstrated the scFv P-9 could suppress the growth of tumor effectively.These findings support the effort to investigate a scFv-based early diagnosis and targeted therapy for prostatic carcinoma. And it also provided a base for the further study.