Construction and application of EV71 ScFv high-throughput cassette expression system

Abstract

Objective To construct a method to express ScFv antibody from PCR products, and use it in phage display for high-throughput ScFv expression. Methods Cytomegalovirus (CMV) promotor, ScFv and BGH-Poly A gene fragments were amplified by PCR. Overlapping PCR was used to form a tandemly linear cassette gene. By transiently transfected into 293T cells, ScFv antibodies expression of cassette gene were tested by Western blot, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA). Ninety-six clones of antibody genes in phage library were selected and expressed by cassette expression system. The expression level was evaluated and analyzed. Results Three fragments were obtained and a cassette expression system formed. Cassette expression system worked successfully in 293T cells, as a Mr.38×103 brand could observed in Western blot assay. The expressed antibody could specifically bind to its antigen by result of ELISA and IFA. This cassette expression system could also be used in phage display for high-throughput panning. Conclusions The cassette expression system was constructed successfully and high-throughput antibody expression has been achieved. Key words: ScFv antibody; Antibody cassette; Overlapping PCR; High-throughput expression