人BLyS免疫抑制分子的克隆、表达及纯化

Cloning, expression, and purification of the immune inhibitor of human BLyS

目的用异源性T辅助细胞表位修饰人可溶性BLyS突变体(msBLyS),构建和表达BLyS的新型免疫抑制分子。方法经PCR方法构建msBLyScDNA,然后分别与鸡卵清溶菌酶(HEL)或卵清蛋白(OVA)Th表位DNA序列重组,行序列测定后,构建于高效原核表达载体pQE-80L并转化E.coliDH5α,经IPTG诱导表达,Ni-NTA层析纯化后,通过细胞增殖实验检测融合蛋白的生物学活性。结果DNA测序表明,重组HELmsBLyS和OVAmsBLyS的DNA序列与文献报道的序列完全一致。HELms-BLyS和OVAmsBLyS在大肠杆菌中以可溶性形式高效表达。经Ni-NTA层析纯化后,目的蛋白的纯度可达90%以上。活性检测发现,重组蛋白均不能促进Raji细胞增殖。结论成功构建了BLyS的免疫抑制分子,并获得了高效表达及纯化,纯化产物已丧失了sBLyS所具有的细胞增殖活性,为进一步探讨其治疗作用打下了基础。

Objective To clone and express the immune inhibitors of human BLyS and analyze their biological activities. Methods The mutant soluble BLyS （msBLyS） cDNA was constructed by PCR, and then the mutant cDNA was combined to the DNA sequence of T-helper epitope of hen egg-white lysozyme or ovalbumin respectively. After sequencing, the recombined cDNAs were ligated into the prokaryotic expression vector pQE-80L. With induction of IPTG, the fusion proteins were expressed in E. coli DHSa. The proteins were purified with NiNTA chromatography, and their biological activities were assayed by MTr assay. Results DNA sequencing analysis showed that the sequences of the recombined HELmsBLyS and OVAmsBLyS cDNAs were correct. The recombined proteins were highly expressed as soluble proteins. After purification, the purity of recombined proteins was more than 90%. The biological activity assay showed that the recombined proteins couldn＇t stimulate proliferation of Raji cell. Conclusion The immune inhibitors of human BLyS are successfully constructed. The corresponding proteins are highly expressed and purified, and the purified products lose their biological activities.