异源性Th表位修饰的CTLA4-Ig-msBlys构建及融合蛋白表达

Construction and expression of a recombinant expression protein of CTLA4-Ig-msBlys which modified by Th epitope

目的构建异源性Th表位修饰的CTLA4-Ig-msBlys的原核表达质粒,并在E.coliBL21中诱导融合蛋白表达,初步检测蛋白特性。方法采用PCR法从质粒pCMV-sBlys扩增sBlys片段,将与卵清蛋白(ovalbumin,OVA)Th表位重组后的msBlys连接CTLA4-Ig片段,克隆入pGEX-KG原核表达载体,用LB固体培养基抗生素筛选、酶切并经琼脂糖凝胶电泳鉴定;将质粒pGEX-KG-CTLA4-Ig-msBlys转入E.coliBL21菌株中,实现插入基因的融合表达,用SDS-PAGE和Western-blot检测表达产物。结果 pGEX-KG-CTLA4-Ig-msBlys构建正确且最终转入E.coliBL21,得到融合蛋白的表达;CTLA4-Ig-msBlys融合蛋白在E.coli中获得表达,表达产物的相对分子质量同预期值一致。结论成功构建原核表达质粒pGEX-KG-CTLA4-Ig-msBlys,并在E.coliBL21表达CTLA4-Ig-msBlys融合蛋白,为下一步探讨Blys在自身免疫性疾病患者的治疗作用奠定了基础。

Objective To construct the prokaryotic expression plasmid pGEX-KG-CTLA4-Ig-msBIys and its expression in E. coli BL21-To detect the fusion protein. Methods The DNA fragment of msBlys gene amplified from the plasmid pCMV- sBlys by polymerase chain reaction （PCR）, then connected with OVA and CTLA4-Ig fragment. The DNA fragment of CTLA4- Ig-msBlys was cloned into the prokaryotic expression vector pGEX-KG and expressed as a fusion protein in E. coli BL21 induced by IPTG. The expressed fusion protein CTLA4-Ig-msBlys was purified and identified by SDS-PAGE and Western blot. Results The recombinant expression plasmid pGEX-KG-CTLA4-Ig-msBlys was successfully constructed. The recombinant expression protein was steadily expressed in E. coli, the relative molecular mass of the expression product was identical with the value pre- dicted. Conclusion The recombinant prokaryotic expression plasmid pGEX-KG-CTLA4-Ig-msBlys is constructed successfully transformed into E. coli BL21, then its expressed as a fusion protein CTLA4-Ig-msBlys in E. coli BL21. All that will be helpful for the further study of Blys.