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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 construct and express novel immunosuppressive molecules of BLyS by modifying human soluble BLyS mutant (msBLyS) with heterologous T helper epitopes. Methods The msBLy cDNA was constructed by PCR. The recombinant was cloned into the recombinant plasmid pQE-80L and then transformed into E. coli BL21 (DE3). coli DH5α was induced by IPTG. After purified by Ni-NTA chromatography, the biological activity of the fusion protein was detected by cell proliferation assay. Results DNA sequencing showed that the DNA sequences of recombinant HELmsBLyS and OVAmsBLyS were completely consistent with those reported in the literature. HELms-BLyS and OVAmsBLyS are highly expressed in soluble form in E. coli. Purified by Ni-NTA chromatography, the purity of the target protein up to 90%. The activity test showed that recombinant protein could not promote the proliferation of Raji cells. Conclusion The immunosuppressive molecules of BLyS were successfully constructed and highly expressed and purified. The purified products have lost the cell proliferation activity of sBLyS, which laid the foundation for further study of its therapeutic effect.