非洲猪瘟病毒CD2v蛋白单链抗体的制备及鉴定OA北大核心CSTPCD

To obtain specific single-chain variable fragment(scFv)antibodies against the CD2v protein of African swine fever virus(ASFV),CD2v-positive monoclonal antibody hybridoma cells were used as templates,and its variable regions of the heavy(VH)and light(VL)chains were amplified by PCR,respectively,in this study.The scFv genes were constructed through insertion of short peptide linker(Gly4Ser)2 be-tween VH and VL by Overlap-PCR.The scFv genes were inserted into the prokaryotic expression vector pET28a(+),which was transformed into Escherichia coli BL21(DE3).The scFv protein expression was induced with 1 mmol/L IPTG at 25 ℃,then scFv reactivity was evaluated by ELISA.Results showed that the antibod-ies hybridoma secreted by cell line 4B8 could effectively block antibodies from pig sera against CD2v protein in ELISA.And 4B8-scFv gene(744 bp)was successfully constructed by joining VH(399 bp)and VL(369 bp)of a monoclonal antibody(mAb)via a GS linker.SDS-PAGE result revealed that 4B8-scFv was expressed as inclusion bodies in E.coli BL21(DE3).High-purity 4B8-scFv(34 ku)was produced in inclusion bodies,which were further solubilized in urea denaturation with urea and refolding.Indirect ELISA results demonstrated good reactivity of 4B8-scFv with the CD2v protein.