Recovery of IgG binding capability of human FcγRⅡa refolded by rapid dilution expressed in E. coli / 中华微生物学和免疫学杂志

ABSTRACT

Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.