Expression of the fusion protein of human soluble B lymphocyte stimulator and diphtheria toxin in E.coli and its biological activity

Objective To prepare the fusion protein of human soluble B lymphocyte stimulator and diphtheria toxin(hsBAFF-DT388) in E.coli and investigate its targeted activity to human B cells.Methods The hsBAFF-DT388 gene was optimized in advance and inserted into the clone vector pMD19-T.The fragment of the hsBAFF-DT388 fusion gene was separated from the plasmid pUC57-hsBAFF-DT388 by PCR according to hsBAFF-DT388 gene order.The recombinant was screened with colony PCR,restriction map analysis and DNA sequencing.The recombinant vector was digested by restriction enzymes,and then inserted into the expression vector pColdⅡ.The positive recombinant expression vector was identified by colony PCR and restriction map analysis.The recombinant strain was induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blot,and then purified by Ni2+-NTA affinity chromatography.Biological activity of the purified protein was detected by cell fluoresce and MTT assay.Results Expression level of the recombinant protein accounted for 40% of total bacterial proteins of E.coli,and the recombinant protein could bind with BAFF receptor-positive cells and had a cytotoxic effect on human B cells(Hmy2.CIR).Conclusion The fusion protein expression vector of hsBAFF-DT388 was successfully constructed and the recombinant protein with selective cytotoxicity against B cell was obtained,which may establish a solid foundation for further study of the therapy for B cell malignancies and autoimmune diseases.