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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。
OBJECTIVE: To construct the recombinant expression vector PQE30-tBID using PQE-30 as a prokaryotic expression vector and express and purify the target protein tBID. Anti-human HER2 antibody was conjugated with tBID protein to anti-HER2-Gold Mag -tBID to explore its pro-apoptotic effect on prostate cancer cells. Methods: The specific upstream and downstream primers were designed according to the gene sequence of tBID. The target gene tBID was amplified by general PCR. The recombinant expression vector PQE30-tBID was transformed into BL21 (DE3) and induced by IPTG. SDS- Gel electrophoresis and Western Blot identification analysis to verify the expression of the target protein tBID, and its purification. The human HER2 antibody and the tBID protein were coupled to the surface by the electrostatic interaction between the gold nanoparticles and the protein and the hydrophobic interaction to construct the molecular probe Anti HER2-Gold Mag-tBID. Flow cytometry was used to detect the specific affinity between the molecular probe and prostate cancer PC-3 cells. Annexin V-FITC apoptosis assay kit was used to analyze the pro-apoptotic effect of molecular probes on PC-3 cells. Results: The 411 bp DNA fragment was obtained after normal PCR amplification. The double digestion and sequencing showed that the recombinant expression vector PQE30-tBID was successfully constructed. The pro-apoptotic protein tBID was successfully expressed in E. coli. The relative molecular weight of the protein was about 15 KD. After purification, tBID protein was obtained with high purity. After the coupling with gold nanoparticles, a new type of molecular probe Anti HER2-Gold Mag-tBID was successfully constructed. The molecular probe could specifically bind to PC-3 cells, and apoptosis of PC-3 cells was observed by Annexin V-FITC staining. The apoptotic rate was 62.9%, which was significantly higher than that in untreated group (3.79%) and control group (4.33%), there is a significant statistical difference. CONCLUSION: PQE30-tBID recombinant expression vector can be highly expressed in E. coli, and the highly purified human tBID protein has been successfully obtained. Through the coupling of gold nanoparticles, this protein can be reconstituted with human HER2 antibody into a novel molecular probe and can specifically target prostate cancer PC-3 cells and promote its apoptosis significantly.