中文摘要：

目的：以PQE-30为原核表达载体,构建PQE30-tBID重组表达载体,表达和纯化目的蛋白tBID,并利用金磁纳米微粒将tBID蛋白与人HER2抗体偶联成分子探针Anti HER2-Gold Mag-tBID,以探究其对前列腺癌细胞的促凋亡作用。方法：根据tBID的基因序列设计特异性的上下游引物,利用普通PCR扩增目的基因tBID,构建重组表达载体PQE30-tBID,将其转化到BL21（DE3）中,IPTG诱导表达,经SDS-PAGE凝胶电泳和Western Blot鉴定分析,验证目的蛋白tBID的表达,并对其进行纯化。利用金磁纳米微粒与蛋白质之间的静电相互作用以及疏水相互作用,将人HER2抗体与tBID蛋白偶联在其表面,构建分子探针Anti HER2-Gold Mag-tBID。流式细胞术检测该分子探针与前列腺癌PC-3细胞的特异性亲附结合能力,通过Annexin V-FITC细胞凋亡检测试剂盒分析分子探针对PC-3细胞的促凋亡作用。结果：普通PCR扩增后得到了411 bp的DNA片段,经双酶切鉴定以及菌液测序,表明重组表达载体PQE30-tBID构建成功。促凋亡蛋白tBID成功地在大肠杆菌中表达,蛋白相对分子量约15 KD,经过纯化,得到了纯度较高的tBID蛋白。经过与金磁纳米微粒的偶联,成功构建出一种新型的分子探针Anti HER2-Gold Mag-tBID。该分子探针可与PC-3细胞特异性结合,且经Annexin V-FITC染色分析可见PC-3细胞发生明显凋亡,凋亡率达62.9%,与未处理组（3.79%）和对照组（4.33%）相比,具有显著的统计学差异。结论：PQE30-tBID重组表达载体能在大肠杆菌中高效表达,且成功得到了纯度较高的人促凋亡蛋白tBID。经金磁纳米微粒偶联,该蛋白能够与人HER2抗体重组成新型的分子探针,且能特异性地靶向前列腺癌PC-3细胞并显著促进其凋亡。

英文摘要：

Objective： To construct the recombinant plasmid PQE30-tBID,express and purify proapoptosis protein tBID,and further construct the molecular probe Anti HER2-Gold Mag-tBID through combining tBID protein with Gold Mag-nanoparticles,and finally study the specific proapoptosis ability of the molecular probe to PC-3 cell.Methods： The upstream primer and downstream primer were respectively designed to amplify tBID gene segment through Polymerase Chain Reaction（PCR）,and then the recombinant plasmid PQE30-tBID was constructed.Transform this plasmid into E.coli BL21（DE3） to see if the tBID protein was induced to express by using Isopropyl-β-D-thiogalactopyranoside（IPTG）.The expression of the targeted protein was evaluated by SDS-PAGE and Western Blot analysis,and finally,the protein was purified and obtained by Nickel column purification.Then the tBID protein was linked with anti-HER2 antibody by Gold Mag-nanoparticles and finally a molecular probe Anti HER2-Gold Mag-tBID was constructed.The specific binding of the molecular probe to prostate cancer cells PC-3 was evaluated by FCM and Annexin V-FITC staining analysis was applied to see if the molecular probe could promote PC-3 cell to apoptosis.Results： The targeted DNA fragment,showing a length of 411 bp in agarose gel electrophoresis（AGE）,was obtained through PCR.Restrictive enzyme digestion and DNA sequencing showed the recombinant plasmid PQE30-tBID was constructed successfully.The proapoptosis protein tBID was expressed in E.coli correctly with a relative molecular weight of about 15 KD.The purity of protein was analyzed and reached high after purification.In addition,the novel molecular probe Anti HER2-Gold Mag-tBID was constructed successfully.The probe could bind with HER2 in the PC-3 cell membrane specifically and induce the PC-3 cells to apoptosis.The apoptosis rate reached to 62.9 %,which was significantly higher than that of the untreated group and the control group.Conclusion： The recombinant plasmid PQE30-tBID could be expressed in E.