人转化生长因子β1RNA干扰真核表达载体的构建

发布时间：2018-03-13 08:48

  本文选题：转化生长因子β1(TGF-β1)　切入点：RNA干扰(RNAi)　出处：《昆明医学院》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：构建针对人转化生长因子β1(transforming growth factor beta 1，TGF-β1)的RNA干扰(RNA interference，RNAi)真核表达质粒载体，，为进一步研究RNAi对纤维化疾病的治疗作用奠定基础。 方法：应用生物信息学技术从GeneBank上查找到人TGF-β1 cDNA序列，参考siRNA的设计原则，成功设计了3条针对人TGFβ1基因的不同序列的siRNA及其相应的阴性对照siRNA。成功设计并合成了上述siRNA相应的shRNA的编码DNA，将其分别退火形成shRNA双链并与psiSTRIKE Neomycin载体连接。将连接产物转化大肠杆菌(E. coli)DH5α，提取质粒，经PstⅠ酶切鉴定筛选出重组质粒，DNA测序分析DNA插入片段的序列。 结果：经BLAST序列比对证实3条siRNA与人TGF-1基因同源而与其它基因无同源性，3条阴性对照siRNA与人类基因组DNA无序列同源性。重组载体经PstⅠ酶切产生958bp和3655bp两条带，与预期结果相符。基因测序证实DNA插入片段序列与所设计的shRNA编码DNA序列完全一致。 结论：成功构建了针对人TGF-β1基因的siRNA真核表达载体及其阴性对照siRNA真核表达载体，从而为进一步研究RNAi对纤维化疾病的治疗作用奠定基础。
[Abstract]:Objective: to construct the eukaryotic expression plasmid vector targeting human transforming growth factor beta 1 (TGF- 尾 1) of human transforming growth factor beta 1 (TGF- 尾 1), so as to lay a foundation for further study on the therapeutic effect of RNAi on fibrosis disease. Methods: the sequence of human TGF- 尾 1 cDNA was found from GeneBank by bioinformatics, and the design principle of siRNA was consulted. Three siRNA fragments targeting different sequences of human TGF 尾 1 gene and their corresponding negative control siRNAs were successfully designed and synthesized. The corresponding shRNA coding DNAs of the siRNA were successfully designed and synthesized, and annealed to form shRNA double strand respectively and connected with psiSTRIKE Neomycin vector. The ligation product was transformed into E. coli)DH5 伪 and the plasmid was extracted. The recombinant plasmids were sequenced by Pst 鈪

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