Abstract:

【Objective】To construct the gene of mouse anti-Xac disulfide stabilized Fv fragments (dsFv), express and refold to the form of dsFv with biological activity.【Method】The genes of VH and VL raised against Xac were mutated by the method of PCR-based mutagenesis, and then cloned into expression plasmid. The recombinant plasmids were transformed into E. coli BL21 (DE3) strain and these two genes were expressed. The inclusion body proteins of VH and VL were harvested and dissolved, and then diluted into refolding solution pro rata to form dsFv antibody which was further purified by HisTrap HP column. The products of expression and renaturation were analyzed by SDS-PAGE and western-blot. The affinity of dsFv to Xac-LPS was determined by BIAcore. The specificity and stability were detected by ELISA.【Result】 Sequence analysis proved that cysteines were introduced into VH44 and VL100, and the genes expressed in E.coli BL21 (DE3). Most of the protein existed in the form of inclusion body, the expression products were refolded successfully. SDS-PAGE showed that the molecular masses were 23 kD for VH and VL, and 46 kD for dsFv. BIAcore analysis showed that dsFv retained high affinity to Xac-LPS with an affinity constant (KD) of 3.40×10-10M. ELISA indicated that dsFv had a high specificity to Xac. Compared with scFv’s, the thermal stability of dsFv elevated nearly 20℃.【Conclusion】dsFv was expressed and refolded successfully, which maintained high affinity, specificity and stability. This research established a solid basis for rapid diagnosis of Xac.

Key words: Xac')">Xac, dsFv, inclusion body, renaturation, BIAcore analysis