链霉亲和素-beclin1重组载体的构建以及融合蛋白的原核表达和纯化

发布时间：2018-03-03 10:19

  本文选题：前列腺癌　切入点：自噬　出处：《第四军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：前列腺癌（prostate cancer，PCa）是一种老年男性泌尿系统的疾病，随着生活水平和诊疗手段的不断提高以及我国人口进入老龄化，前列腺癌发病率逐年增高。但是目前的医疗水平，使前列腺癌确诊时基本已是中晚期，而且传统的治疗手段如手术治疗、激素治疗等疗效欠佳，放化疗等治疗方法由于缺乏靶细胞的特异性，对正常的身体组织也会产生严重的破坏，在临床应用上受到一定的限制，克服化疗药物的副作用，提高杀灭癌细胞的特异性，是目前比较重要的研究课题之一。 激素非依赖性前列腺癌自噬作用缺失、抗凋亡能力增强可能是其肿瘤细胞增殖的原因。自噬相关基因beclin1是目前新发现的抑癌基因，源于核孤儿受体的小肽NuBCP-9可以促进细胞凋亡，前列腺特异性膜抗原(prostate-specific membraneantigen，PSMA)的适配子能特异地与前列腺癌细胞结合。本课题拟利用链霉亲和素-生物素技术将beclin1与小肽NuBCP-9以及PSMA适配子偶联在一起，特异地增强前列腺癌细胞的自噬和凋亡，试图为前列腺癌的治疗寻找一条有效的新途径。 1、构建SA-beclin1原核表达载体 从GenBank中检索出核心链霉亲和素Core SA基因的DNA序列后，，利用in-Fusion引物设计软件设计含有BamHⅠ酶切位点的SA的特异性引物。以Core SA作为模板，利用PCR技术扩增出SA，长度为381bp。自噬相关基因beclin1已在前期工作中构建入原核表达载体pQE80L，用in-Fusion方法连接PCR产物SA和pQE80L-beclin1，酶切鉴定并送公司测序。SA-beclin1测序结果与预期完全一致，重组载体构建成功，命名为pQE80L-SA-beclin1。2、融合蛋白SA-beclin1的原核表达与纯化 将重组载体pQE80-SA-beclin1转化入大肠杆菌Rosetta-gami原核表达菌，用IPTG诱导表达融合蛋白SA-beclin1，通过优化表达条件，选择37℃，IPTG浓度0.5mmol/L，诱导5h。用镍亲和凝胶层析柱纯化融合蛋白，Western blot鉴定融合蛋白表达情况。结果显示IPTG诱导后SA-beclin1融合蛋白(含His标签)在大肠杆菌中高效表达。SDS-PAGE分析表达的融合蛋白以包涵体为主，经镍亲和凝胶层析柱纯化得到融合蛋白，Western blot鉴定其相对分子量约为72000，与预期相符。包涵体经变性复性后，亦可得到与目的条带大小相符的蛋白。 设计并合成小肽NuBCP-9，并分别标记生物素和FITC，得到纯度90%的生物素标记小肽NuBCP-9，为后续实验提供基础。 以上结果表明，成功构建了重组质粒并表达纯化了SA-beclin1融合蛋白，得到生物素标记的小肽NuBCP-9，为进一步研究SA-beclin1的功能及临床应用奠定了基础。
[Abstract]:Prostate cancer (PCA) is a disease of the urinary system in elderly men. With the improvement of living standard and diagnosis and treatment means and the aging of population in our country, the incidence of prostate cancer is increasing year by year. The diagnosis of prostate cancer is basically late, and the traditional treatment methods, such as surgery, hormone therapy and so on, are not good. The radiotherapy and chemotherapy methods lack the specificity of target cells. It is one of the most important research topics to overcome the side effects of chemotherapeutic drugs and to improve the specificity of killing cancer cells. The loss of autophagy and the increase of anti-apoptotic ability of steroid independent prostate cancer may be the reasons for the proliferation of tumor cells. Autophagy associated gene beclin1 is a newly discovered tumor suppressor gene. Small peptide NuBCP-9, derived from nuclear orphan receptors, promotes apoptosis. The aptamers of prostate-specific membrane antigens (PSMA) can specifically bind to prostate cancer cells. In this study, beclin1 was coupled with small peptide NuBCP-9 and PSMA aptamers by streptavidin-biotin technique. It specifically enhances autophagy and apoptosis of prostate cancer cells, and attempts to find a new effective way for the treatment of prostate cancer. 1. Construction of prokaryotic expression vector of SA-beclin1. The DNA sequence of core streptavidin Core SA gene was retrieved from GenBank. The specific primers of SA containing BamH 鈪

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