Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.
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 DH5alpha 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.
Gao
Huiguang
H
Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing , 400038, PR China.
Fu
Weiling
W
Li
Rongfen
R
Chen
Linfeng
L
Ji
Qing
Q
Zhang
Li
L
Huang
Gang
G
He
Fengtian
F
D000818:Animals; D000917:Antibody Formation; D053264:B-Cell Activating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D049109:Cell Proliferation; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010047:Ovalbumin / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*
D053264:B-Cell Activating Factor; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D010047:Ovalbumin