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目的构建携带跨膜信号序列的凋亡蛋白(apoptin)的原核表达载体PET-28a(+)-apoptin,诱导表达并纯化重组apoptin蛋白,制备多克隆抗体。方法用多重PCR的方法扩增跨膜信号序列(PTD)及apoptin的编码序列,构建原核表达载体PET-28a(+)-PTD-apoptin,转化大肠杆菌BL21(DE3),低温条件下IPTG诱导目的基因表达,SDS-PAGE方法鉴定蛋白表达,经镍亲和层析方法纯化目的蛋白后,免疫BALB/c小鼠,制备多克隆抗体。结果 DNA测序鉴定表明成功构建了原核表达载体PET-28a(+)-PTD-apoptin,该重组质粒转化大肠杆菌BL21(DE3),经低温和IPTG诱导后,获得重组apoptin蛋白,制备的多克隆抗体经ELISA方法和免疫印迹法证实能特异性地识别apoptin。结论成功获得了携带跨膜信号序列的重组蛋白apoptin及其多克隆抗体,为进一步研究其特异性诱导肿瘤细胞凋亡功能及相关机制奠定了基础。
Objective To construct the prokaryotic expression vector PET-28a (+) - apoptin carrying the transmembrane signal sequence of apoptin, and to induce the expression and purification of recombinant apoptin protein to prepare polyclonal antibody. Methods The coding sequence of transmembrane signal sequence (PTD) and apoptin were amplified by multiplex PCR. The prokaryotic expression vector PET-28a (+) - PTD-apoptin was constructed and transformed into E.coli BL21 (DE3) The protein expression was identified by SDS-PAGE and the protein of interest was purified by nickel affinity chromatography. BALB / c mice were immunized to prepare polyclonal antibody. Results DNA sequencing showed that the prokaryotic expression vector PET-28a (+) - PTD-apoptin was successfully constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3). After induced by low temperature and IPTG, recombinant apoptin protein was obtained. The prepared polyclonal antibody Confirmed by ELISA and Western blot can specifically identify apoptin. Conclusion The recombinant protein apoptin carrying the transmembrane signal sequence and its polyclonal antibody were successfully obtained, which laid the foundation for further study on its function of inducing tumor cell apoptosis and its related mechanisms.