Cloning, Expression and Activity Determination of the Recombinant Human Soluble B Lymphocyte Stimulator and Its Two Mutants

( 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. The
two mutants of hsBLyS: hsBY-A (Cys¹⁴⁶→Ala¹⁴⁶,
TGC→GCT) and hsBY-V (Cys¹⁴⁶ →Val¹⁴⁶, TGC→GTT),
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 DH5α 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.