[Inhibitory effect of novel internalized fully human phage antibody fragments on proliferation of lung adenocarcinoma cell line overexpressing peroxiredoxin I].

Abstract

Background and objective: Previous researches have implicated the close relationship between peroxiredoxin I (Prx I) and cancer progression. A lung adenocarcinoma-related human phage antibody library has been constructed by using phage display techniques. This study was to screen out the single chain variable fragment (scFv) antibodies from the library against a lung adenocarcinoma cell line overexpressing Prx I and to analyze their anti-proliferation ability.

Methods: The insertion ratio of scFv gene was identified by polymerase chain reaction (PCR). The products were digested by Sfi I and Not I, and analyzed on 1% agarose gel. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed separately, and the positive clones were chosen for soluble expression. The internalization of radiolabeled scFv fragments was then quantified. The proliferation and apoptosis of A549 cells were detected by MTT assay and flow cytometry (FCM). The protein expression of Prx I in A549 cells was analyzed by Western blot.

Results: The insertion ratio of scFv gene was 77% (23/30) and enzyme digestion showed the target products. The sixth phage harvest yielded 180 times as much as that of the first one. Positive reactions with A549 cells were detected in six (60%) of ten random clones. The human scFv fragments against Prx I of lung adenocarcinoma were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). The internalized scFvs mediated cell apoptosis and Prx I expression down-regulation.

Conclusions: The scFv fragments against Prx I of lung adenocarcinoma are acquired by screening the phage antibody library. The soluble antibodies have specific avidity and inhibitory effect on proliferation of human lung adenocarcinoma cells.