靶向MRP1基因重组质粒表达载体构建医学毕业设计报告.doc

齐 齐 哈 尔 大 学 毕业设计（论文） 题 目 靶向MRP1基因pRNAT-H1.1/shuttle-RFP重组质 粒表达载体构建 学 院 专业班级 学生姓名 指导教师 成 绩 2011 年 6 月 20 日  摘 要 癌症严重威胁着人类的健康，其发病率呈上升趋势。化疗作为其常规临床治疗手段，在癌症治疗中具有手术和放射治疗不能替代的作用。肿瘤细胞的多药耐药性（multidrug resistance, MDR）是导致肿瘤细胞化疗失败的主要原因。肿瘤细胞产生多药耐药的原因较为复杂，多药耐药相关蛋白1（Multidrug Resistance-associated Protein 1，MRP1）的过度表达是导致其产生多药耐药的主要原因之一。RNA干扰(RNA interference，RNAi)是近年来发现的能快速、高效、特异的沉默目的基因表达的技术，如能通过RNAi技术沉默MDR1基因，逆转肿瘤细胞的多药耐药性将为改善癌症病人的化疗效果奠定基础。 目的：本课题选用pRNAT-H1.1/shuttle-RFP表达穿梭载体。构建针对mrp1 mRNA的RNA干扰表达载体。 方法：将预先根据MRP1基因序列设计合成的编码siRNA的cDNA序列与pRNAT-H1.1/shuttle-RFP质粒载体连接，构建靶向mrp1 siRNA重组质粒。将重组质粒转化E.coli DH5α后大量提取重组质粒，经菌落 PCR和 DNA测序分析检测重组载体构建结果。 结果：成功构建靶向MRP1基因pRNAT-H1.1/shuttle-RFP重组质粒表达载体。为下一步抑制mrp1基因在肿瘤细胞中的表达奠定基础。 关键词：RNA干扰；MRP1；pRNAT-H1.1/shuttle-RFP质粒；穿梭载体 Abstract Cancer, a serious threat to human health, the incidence are on the rising trend. Chemotherapy as the routine clinical therapy of tumor, which is an irreplaceable way in cancer treatment. MDR of tumor cell cause the failure of chemotherapy treatment . The reason causing the cancer MDR is relatively complicated, and the excessively expression of multidrug resistance-associated protein 1(MRP1) is one of the reasons causing MDR. Silencing MDR1 gene by RNAi, we can improve the effect of chemotherapy by decrease the level of MDR. Objective: In this study, pRNAT-H1.1/shuttle-RFP plasmid was selected to construct the RNAi expression vector of mrp1 mRNA. Methods: According to mrp1 we designed and synthesized the DNA fragment that was cloned into pRNAT-H1.1/shuttle-RFP vector to construct recombinant vector. Transformed the recombinant vector into E.coli DH5α. The result of recombinant vector was analyzed and identified using PCR and DNA sequencing . Results: Constructing pRNAT-H1.1/shuttle-RFP recombinant plasmid expression vector was constructed succe