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[Clonging, expression and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants]. | LitMetric

[Clonging, expression and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants].

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)

Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.

Published: November 2002

AI Article Synopsis

  • BLyS is a protein that helps B cells grow, and researchers amplified its cDNA from human blood samples to study it further.
  • They created two mutant versions of the protein, modifying a specific amino acid, and formed new recombinant plasmids to express these proteins in E. coli bacteria.
  • The resulting proteins were purified and tested, showing high activity in promoting the survival and proliferation of B cells, with one mutant proving to be even more effective than the original version.

Article Abstract

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.

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