曼氏迭宫绦虫翻译控制肿瘤蛋白(TCTP)的原核表达、纯化及其免疫原性分析

Prokaryotic expression,purification,and analysis of the immunogenicity of translationally controlled tumor protein(TCTP)from Spirometra mansoni

目的原核表达、纯化曼氏迭宫绦虫翻译控制肿瘤蛋白(Sm TCTP),并分析其免疫原性。方法构建曼氏迭宫绦虫翻译控制肿瘤蛋白重组表达质粒pGEX-4T-1-Sm TCTP,转化大肠埃希菌BL21菌株后用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达TCTP蛋白,用GST树脂纯化表达产物,并进行SDS-PAGE电泳分析,通过Western blot鉴定重组蛋白的免疫原性。结果 PCR、双酶切、测序结果均表明pGEX-4T-1-Sm TCTP重组质粒构建成功,并在大肠埃希菌中表达可溶性目的蛋白,经GST树脂纯化后获得高纯度的重组目的蛋白。SDS-PAGE分析表达产物为GST融合蛋白,分子质量单位为45ku,与Sm TCTP和GST融合蛋白理论分子质量相符。Western blot显示重组融合蛋白能被相应大鼠抗血清识别。结论成功进行了Sm TCTP的原核表达并获得纯化的重组蛋白,重组蛋白具有良好的免疫原性,为研究Sm TCTP的功能奠定了基础。

Objectives To express Sm TCTP in a prokaryotic expression system,purify the recombinant protein,and analyze its immunogenicity. Methods The Sm TCTP recombinant expression plasmid pGEX-4T-1-Sm TCTP was constructed and transformed into Escherichia coli BL21 cells.Expression of the plasmid was induced with isopropyl-β-D-1-thiogalactopyranoside（IPTG）,and the expression products were purified with GST resin and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）.The immunogenicity of the recombinant protein was determined with Western blotting. Result PCR,double enzyme digestion,and DNA sequencing confirmed that the pGEX-4T-1-Sm TCTP recombinant plasmid was successfully constructed.A soluble target protein was expressed in Escherichia coli,and the recombinant protein was obtained by GST resin purification.SDS-PAGE results indicated that the expression product was a GST fusion protein with a molecular weight of about 45 ku,which coincides with the theoretical molecular weight of an Sm TCTP GST fusion protein.Results of Western blotting indicated that the recombinant fusion protein was recognized by specific antibodies in SD rats. Conclusion Recombinant Sm TCTP was successfully obtained,and this recombinant protein was highly immunogenic.This work provides a basis for the functional study of Sm TCTP.