title: Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli.
abstract: Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.
authors.affiliation: College of Life Sciences, Suzhou University, Suzhou, People's Republic of China.
authors.forename: Fengfeng
authors.initials: F
authors.lastname: Zheng
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Qiu
authors.forename: Yongjing
authors.initials: Y
authors.lastname: Chen
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.forename: Huating
authors.initials: H
authors.lastname: Zhu
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhu