[Clonging, expression and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants].
B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily that functionally involved in B cell proliferation. Here, the full length cDNA of human soluble BlyS (hsBLyS) was amplified by reverse transcription-PCR from total RNA of human peripheral blood lymphocytes and sequenced. The cloned cDNA fragments was inserted into pBV220, a thermo-sensitive expression plasmid, forming recombinant plasmid pBV220/hsBLyS. The two mutants of hsBLyS: hsBY-A (Cys(146) right curved arrow Ala(146), TGC right curved arrow GCT) and hsBY-V (Cys(146) right curved arrow Val(146), TGC right curved arrow 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 DH5alpha 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 products were identical with the published hsBLyS cDNA sequence or expected mutant sequence 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.
Chen
Guang-Yu
GY
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. wangjx@nic.bmi.ac.cn
Zou
Min-Ji
MJ
Peng
Shan-Yun
SY
Wang
Jia-Xi
JX
D053264:B-Cell Activating Factor; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D053264:B-Cell Activating Factor; D008565:Membrane Proteins; D011994:Recombinant Proteins; C505719:TNFSF13B protein, human; D014409:Tumor Necrosis Factor-alpha