( Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China )

Abstract

B lymphocyte stimulator(BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily that functionally involved in B cell proliferation. Her e, the full length cDNA of human soluble BlyS(hsBLyS) was amplified by reverse t ranscription PCR from total RNA of human peripheral blood lymphocytes and sequenced. The cloned cDNA fragments was inserted into pBV220, a thermo-sensitive ex pr ession plasmid, forming recombinant plasmid pBV220/hsBLyS.ª« The two mutants of hsBLyS: hsBY-A (Cys¹⁴⁶¡úAla¹⁴⁶, TGC¡úGCT) and hsBY-V (Cys¹⁴⁶ ¡úVal¹⁴⁶, TGC¡úGTT), were amplified by overlap PCR from pBV220/hsBLyS and inserted into the pBV220 to form pBV220/hsBY-A and pBV220/hsBY-V expression plasmids as the method above. The three expression plasmids were transformed into E.coli DH5¦Á and it was found that the recombinant proteins were highly expressed as inclusion bodies. Pure recombinant proteins were obtained by Sephacryl-200 gel filtration and renaturation. The experimental results demonstrated that the sequences of the PCR product s were identical with the published hsBLyS DNA sequence or expected mutant sequ ence and the purity of the recombinant proteins obtained was relatively high. The activity of the purified recombinant proteins was very significant in the B cell survival and proliferation assay, and statistical tests indicated that the mutant rhsBY-V promoted proliferation of B lymphocyte cell remarkably better than the wild type.