| Affiliation: | 1.Faculty of Biomedical Engineering of Zhengzhou University,Zhengzhou,People's Republic of China;2.Emergency Department of Xi’an North Hospital,Xi'an,People's Republic of China;3.Vascular Endocrinology Department of Xijing Hospital Affiliated to the Fourth Military Medical University,Xi'an,People's Republic of China |
| Abstract: | ABSTRACT: We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn[MINUS SIGN]ABO2+CDn[MINUS SIGN]CDO2+BCn[MINUS SIGN]BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity. |
