Studies On Construction Of Anti-ICOSL-scFv-C_H3 Antibody And Its Functional Analysis

The present study aimed to making the humanized renovation for anti-ICOSL-scFv by the scFv fused to the human IgGl CH3 domain. In this way, we could get the fusion protein that had good effect on the treatment of organ transplantation chronic rejection. In our previous study, we constructed the source of anti-ICOSL single-chain Fv library. The anti-ICOSL svFv was selected by using ribosome display technology from the scFv library. But the scFv is cleared rapidly in vivo and easy to be cleared in the blood in the absence of Fc fragment. In order to compensate for this defect, we connected the anti-ICOSL-scFv with CH3 gene for the anti-ICOSL antibody humanized. The detailed results were summarized as follows:1 Generation of humanized anti-ICOSL-scFv-CH3 and construction of expression vector of pET43.1a/scFv-CH3The total RNA was isolated from mononuclear cells of human peripheral blood. The IgGl CH3 gene was amplified by reverse transcription-PCR (RT-PCR). Finally, the CH3 gene was connected with anti-ICOSL-svFv by SOE-PCR. The results showed that the correct anti-ICOSL-scFv-CH3 gene sequence was obtained as we expectation by gel electrophoresis and gene sequencing identification.2 Expression, purification and identification of the anti-ICOSL-scFv-CH3 antibodyThe fusion gene was inserted into vector pET43.1a and expressed in BL21 (DE3) E. coli. The fusion protein was purified by metal chelate affinity chromatography (Ni-NTA) and obtained high purity of antibody protein. And we identified its antigen specific binding by ELISA and Western blotting. The results showed that we built the anti-ICOSL-scFv-CH3 expression vector and it had specific binding with ICOSL antigen.3 Functional analysis of humanized anti-ICOSL-scFv-CH3 in vivoIn order to accurately detected the physiological function of the fusion protein, we took the protein of anti-CD28 and physiological saline as negative control groups, and the anti-ICOSL-scFv-CH3 fusion protein as experiment group. All the groups were orthotopically transplanted in mice tail skin before injection proteins and physiological saline respectively, and then observed the chronic rejection of mice tail skin. The experimental results show that:compared with two control groups, the protein of anti-CD28 and physiological saline, skin graft of the anti-ICOSL-scFv-CH3 protein group were gradually heal over and had new mice hair growing gradually. While the control groups appeared ulceration, scarring and so on. For all of these, the anti-ICOSL-scFv-CH3 fusion protein has significant inhibitory effect on skin allograft rejection. Anti-ICOSL-scFv-CH3 fusion protein provides the basis for the study of treatment of organ transplantation rejection.