携带跨膜信号的凋亡蛋白的原核表达及多克隆抗体的制备

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。

Prokaryotic expression and polyclonal antibody preparation of apoptin carrying protein transduction domain

Objective To construct prokaryotic expression vector PET-28a （＋）-PTD-apoptin for the expression of apoptin carrying protein transduction domain （PTD） and purify recombinant protein for the preparing of polyclonal antibodies. Methods PTD-apoptin coding fragment was obtained by multiplex PCR. Prokaryotic expression vector PET-28a （＋）-PTD- apoptin was constructed and transformed into BL21 （DE3）. The target gene was induced by low temperature and IPTG. The recombinant protein was checked by SDS-PAGE and purified by Ni2＋ affinity chromatography. The polyclonal antibodies were prepared by immunizing BALB/e mice. Results DNA sequencing results indicate that prokaryotic expression vector PET- 28a（＋）-PTD-apoptin was successfully constructed and transformed into BL21 （DE3.）. The recombinant protein was attained by low temperature and IPTG induction. ELISA and Western blot confirmed that prepared polyclonal antibodies could specially recognize apoptin. Conclusion The recombinant protein apoptin carrying PTD and its polyelonal antibodies were prepared, which can be used for the further study on the function and related mechanism of apoptin.