本文選題：轉(zhuǎn)化生長因子β1(TGF-β1)　切入點：RNA干擾(RNAi)　出處：《昆明醫(yī)學(xué)院》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：構(gòu)建針對人轉(zhuǎn)化生長因子β1(transforming growth factor beta 1，TGF-β1)的RNA干擾(RNA interference，RNAi)真核表達質(zhì)粒載體，，為進一步研究RNAi對纖維化疾病的治療作用奠定基礎(chǔ)。 方法：應(yīng)用生物信息學(xué)技術(shù)從GeneBank上查找到人TGF-β1 cDNA序列，參考siRNA的設(shè)計原則，成功設(shè)計了3條針對人TGFβ1基因的不同序列的siRNA及其相應(yīng)的陰性對照siRNA。成功設(shè)計并合成了上述siRNA相應(yīng)的shRNA的編碼DNA，將其分別退火形成shRNA雙鏈并與psiSTRIKE Neomycin載體連接。將連接產(chǎn)物轉(zhuǎn)化大腸桿菌(E. coli)DH5α，提取質(zhì)粒，經(jīng)PstⅠ酶切鑒定篩選出重組質(zhì)粒，DNA測序分析DNA插入片段的序列。 結(jié)果：經(jīng)BLAST序列比對證實3條siRNA與人TGF-1基因同源而與其它基因無同源性，3條陰性對照siRNA與人類基因組DNA無序列同源性。重組載體經(jīng)PstⅠ酶切產(chǎn)生958bp和3655bp兩條帶，與預(yù)期結(jié)果相符�；驕y序證實DNA插入片段序列與所設(shè)計的shRNA編碼DNA序列完全一致。 結(jié)論：成功構(gòu)建了針對人TGF-β1基因的siRNA真核表達載體及其陰性對照siRNA真核表達載體，從而為進一步研究RNAi對纖維化疾病的治療作用奠定基礎(chǔ)。
[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 偽 and the plasmid was extracted. The recombinant plasmids were sequenced by Pst 鈪

本文編號：1605655