中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2010年
11期
1951-1954
,共4页
血管紧张素系统%血管紧张素原%血管紧张素转换酶%小干扰RNA%构建%质粒
血管緊張素繫統%血管緊張素原%血管緊張素轉換酶%小榦擾RNA%構建%質粒
혈관긴장소계통%혈관긴장소원%혈관긴장소전환매%소간우RNA%구건%질립
背景:RNA干扰技术通过将具有一定结构特点、长19～25 bp的双链小干扰RNA导入哺乳动物细胞,特异性降解与其序列具有同源性的mRNA分子,导致目的基因表达抑制.目的:拟构建针对人血管紧张素原mRNA的小干扰RNA表达载体,从而抑制肾素基因在脂肪细胞的表达.方法:从NCBI中查找人血管紧张素原基因全长mRNA序列(NM000029),利用GeneScript公司提供的在线小干扰RNA模板序列设计软件,自行设计靶向血管紧张素原的两条shRNA的DNA模板单链,合成靶向血管紧张素原基因转录可形成茎环结构的寡聚核苷酸,退火后与酶切后的psiRNAT-U6.1/Neo质粒连接,在TOP10菌株中扩增,并测序鉴定.结果与结论:将含有血管紧张素原-mRNA目标序列19 bp的双链DNA插入片段,连接到pRNAT-U6.1/Neo质粒形成重组质粒.EcoR Ⅰ和Hind Ⅲ双酶切后,空载体得到351 bp小片段,而人重组质粒得到397 bp小片段,与预期相符.EcoRI和Kpv Ⅰ双酶切后,空载体得到1条345 bp小片段,而人重组载体没有得到小片段条带,与预期相符.测序结果表明psiRNAT-U6.1/Neo质粒已经插入人脂肪细胞的干扰合成片段,无碱基突变,成功构建了靶向血管紧张素原-小干扰RNA表达载体.
揹景:RNA榦擾技術通過將具有一定結構特點、長19～25 bp的雙鏈小榦擾RNA導入哺乳動物細胞,特異性降解與其序列具有同源性的mRNA分子,導緻目的基因錶達抑製.目的:擬構建針對人血管緊張素原mRNA的小榦擾RNA錶達載體,從而抑製腎素基因在脂肪細胞的錶達.方法:從NCBI中查找人血管緊張素原基因全長mRNA序列(NM000029),利用GeneScript公司提供的在線小榦擾RNA模闆序列設計軟件,自行設計靶嚮血管緊張素原的兩條shRNA的DNA模闆單鏈,閤成靶嚮血管緊張素原基因轉錄可形成莖環結構的寡聚覈苷痠,退火後與酶切後的psiRNAT-U6.1/Neo質粒連接,在TOP10菌株中擴增,併測序鑒定.結果與結論:將含有血管緊張素原-mRNA目標序列19 bp的雙鏈DNA插入片段,連接到pRNAT-U6.1/Neo質粒形成重組質粒.EcoR Ⅰ和Hind Ⅲ雙酶切後,空載體得到351 bp小片段,而人重組質粒得到397 bp小片段,與預期相符.EcoRI和Kpv Ⅰ雙酶切後,空載體得到1條345 bp小片段,而人重組載體沒有得到小片段條帶,與預期相符.測序結果錶明psiRNAT-U6.1/Neo質粒已經插入人脂肪細胞的榦擾閤成片段,無堿基突變,成功構建瞭靶嚮血管緊張素原-小榦擾RNA錶達載體.
배경:RNA간우기술통과장구유일정결구특점、장19～25 bp적쌍련소간우RNA도입포유동물세포,특이성강해여기서렬구유동원성적mRNA분자,도치목적기인표체억제.목적:의구건침대인혈관긴장소원mRNA적소간우RNA표체재체,종이억제신소기인재지방세포적표체.방법:종NCBI중사조인혈관긴장소원기인전장mRNA서렬(NM000029),이용GeneScript공사제공적재선소간우RNA모판서렬설계연건,자행설계파향혈관긴장소원적량조shRNA적DNA모판단련,합성파향혈관긴장소원기인전록가형성경배결구적과취핵감산,퇴화후여매절후적psiRNAT-U6.1/Neo질립련접,재TOP10균주중확증,병측서감정.결과여결론:장함유혈관긴장소원-mRNA목표서렬19 bp적쌍련DNA삽입편단,련접도pRNAT-U6.1/Neo질립형성중조질립.EcoR Ⅰ화Hind Ⅲ쌍매절후,공재체득도351 bp소편단,이인중조질립득도397 bp소편단,여예기상부.EcoRI화Kpv Ⅰ쌍매절후,공재체득도1조345 bp소편단,이인중조재체몰유득도소편단조대,여예기상부.측서결과표명psiRNAT-U6.1/Neo질립이경삽입인지방세포적간우합성편단,무감기돌변,성공구건료파향혈관긴장소원-소간우RNA표체재체.
BACKGROUND:In mammalian cells,introduction of double-stranded small interfering RNA(19-25 bp)can cleave and destroy the cognate RNA,which can result in suppression of gene expression.OBJECTIVE:To construct siRNA expression plasmid for interference angiotensinogen(AGT),thereby,to resist AGT expression in adipose cells.METHODs:The mRNA sequence of AGT gene was searched from NCBI(NM000029).Utilize of GenScript siRNA technology,AGT-siRNA oliaonucletides were chemically synthesized and inserted into pRNAT-U6 1/Neo vector after annealing,then transformed into TOP10.The recombinant plasmid was identified by restriction endonuclease and DNA sequencing.RESULTS AND CONCLUS1ON:The recombinant plasmid psiRNAT-U6.1/Neo-AGT was obtained by connecting 19 bp segment containing AGT-mRNA sequence to pRNAT-U6.1/Neo After EcoR Ⅰ and Hind Ⅲ digestion.351 bp segment was obtained from empty vector.and 397 bp fragment band was obtained form recombinant plasmid,which was coincidence to the expectation.DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pRNAT-U6.1/Neo without base mutation.The interference vector psiRNAT-U6.1/Neo-AGT was successfully constructed.
