人tBID蛋白的表达纯化及其偶联的新型分子探针对前列腺癌细胞的促凋亡作用研究

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。

Prokaryotic Expression and Purification of Human tBID Protein andProapoptosis Ability of Novel Molecular Probe combined with tBID Proteinto Prostate Cancer Cells

Objective:To construct the recombinant plasmid PQE30-tBID, express and purify proapoptosis protein tBID, and furtherconstruct the molecular probe Anti HER2-GoldMag-tBID through combining tBID protein with GoldMag-nanoparticles, and finallystudy the specific proapoptosis ability of the molecular probe to PC-3 cell.Methods:The upstream primer and downstream primer wererespectively designed to amplify tBID gene segment through Polymerase Chain Reaction (PCR), and then the recombinant plasmidPQE30-tBID was constructed. Transform this plasmid into BL21 (DE3) to see if the tBID protein was induced to express by usingIsopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of the targeted protein was evaluated by SDS-PAGE and Western Blotanalysis, and finally, the protein was purified and obtained by Nickel column purification. Then the tBID protein was linked with anti-HER2 antibody by GoldMag-nanoparticles and finally a molecular probe Anti HER2-GoldMag-tBID was constructed. The specificbinding of the molecular probe to prostate cancer cells PC-3 was evaluated by FCMand Annexin V-FITC staining analysis was applied tosee if themolecular probecouldpromote PC-3cell toapoptosis.Results:The targetedDNAfragment, showingalengthof 411bpin agarose gelelectrophoresis (AGE), was obtained through PCR. Restrictive enzyme digestion and DNA sequencing showed the recombinant plasmidPQE30-tBID was constructed successfully. The proapoptosis protein tBID was expressed in 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 probeAnti HER2-GoldMag-tBID was constructed successfully. The probe could bind with HER2 in the PC-3 cell membrane specifically andinduce the PC-3 cells to apoptosis. The apoptosis rate reached to 62.9 %, which was significantly higher than that of the untreated groupand the control group.Conclusion:The recombinant plasmid PQE30-tBID could be expressed in E. coli efficiently and the tBID proteinwas finally purified. Moreover, the novel molecular probe Anti HER2-GoldMag-tBID was constructed successfully with the purifiedtBID protein, which could bind with the prostate cancer cells specifically and lead to the apoptosis of the cells.