翻译调控肿瘤蛋白的原核表达及纯化

Prokaryotic expression and purification of translationally controlled tumor protein

目的原核表达并纯化翻译调控肿瘤蛋白(Translationally controlled tumor protein,TCTP)。方法以人乳腺癌细胞系MCF-7总RNA为模板,PCR扩增TCTP基因,克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。His Trap FF层析柱纯化重组蛋白后,进行Western blot鉴定。结果克隆的目的基因序列正确,未发生碱基突变;重组表达质粒经双酶切鉴定构建正确;表达的重组蛋白相对分子质量约为20 000,主要以包涵体形式表达;纯化后纯度为80%,可与兔抗人TCTP多克隆抗体特异性结合。结论已成功在大肠杆菌BL21(DE3)中表达并纯化了TCTP,为进一步研究其在肿瘤等疾病发生、发展及治疗中的作用奠定了基础。

Objective To express translationally controlled tumor protein（TCTP） in prokaryotic cells and purify the expressed product.Methods TCTP gene was amplified by PCR using the total RNA of human breast cancer MCF-7 cells as template,and cloned into prokaryotic expression vector pET-28a（＋）.The constructed recombinant plasmid pET28a-TCTP was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was purified by His Trap FF chromatography and identified by Western blot.Results The sequence of cloned TCTP gene was correct,in which no base mutation was observed.Restriction analysis showed that recombinant plasmid pET28a-TCTP was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 20 000,mainly existed in a form of inclusion body and reached a purity of 80% after purification,which showed specific binding to rabbit anti-human polyclonal antibody against TCTP.Conclusion TCTP was successfully expressed in E.coli BL21（DE3） and purified,which laid a foundation of further study on the role of TCTP in the onset,progress and treatment of tumor and other diseases.