Gao Huiguang, Fu Weiling, Li Rongfen, Chen Linfeng, Ji Qing, Zhang Li, Huang Gang, He Fengtian
Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing , 400038, PR China.
Biotechnol Lett. 2006 Oct;28(20):1649-54. doi: 10.1007/s10529-006-9139-y. Epub 2006 Aug 11.
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.
构建了编码可溶性B淋巴细胞刺激因子（134 - 285个氨基酸，sBLyS）突变体的DNA，其中217 - 224位残基被两个甘氨酸取代（命名为msBLyS）。然后将编码来自卵清蛋白（OVA）的外源免疫显性T辅助表位的序列连接到msBLyS cDNA的5'端。测序后，将重组DNA连接到原核表达载体pQE - 80L中。用IPTG诱导后，在大肠杆菌DH5α中产生重组蛋白，其产量占细菌总蛋白的40%以上。用镍 - 次氮基三乙酸（Ni - NTA）层析和Sepharcryl S200层析纯化重组蛋白，使其纯度超过98%。用重组蛋白免疫BALB/c小鼠，可高水平产生抗BLyS抗体，这表明用T辅助表位修饰的重组BLyS突变体在体内能引发与BLyS具有交叉反应性的多克隆抗体。因此，这种重组蛋白可用作BLyS的免疫抑制剂，用于治疗与BLyS相关的自身免疫性疾病。
