1. Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.
- Author
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Huiguang Gao, Weiling Fu, Rongfen Li, Linfeng Chen, Qing Ji, Li Zhang, Gang Huang, and Fengtian He
- Subjects
DNA ,PROTEINS ,GENETIC recombination ,RECOMBINANT proteins ,B cells ,GENES - Abstract
The DNA encoding soluble B lymphocyte stimulator (134–285 amino acids, sBLyS) mutant with residues 217–224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5′-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5α after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo . This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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