哈尔滨医科大学学报
哈爾濱醫科大學學報
합이빈의과대학학보
JOURNAL OF HARBIN MEDICAL UNIVERSITY
2010年
1期
19-21,25
,共4页
基质金属蛋白酶-9%RNA干扰%慢病毒载体
基質金屬蛋白酶-9%RNA榦擾%慢病毒載體
기질금속단백매-9%RNA간우%만병독재체
matrix metalloproteinase-9%RNA interference%lentiviral vector
目的 构建小鼠基质金属蛋白酶-9(matrix metalloproteI_(Na)se-9,MMP-9)RNA干扰(RNA interference,RNAi)慢病毒载体.方法 针对小鼠MMP-9基因序列,利用公用网站按照RNAi序列设计原则,设计RNAi靶点序列,并合成靶序列的Oligo DNA,退火形成双链DNA,与经HpaⅠ和XhoⅠ酶切后的pGCL-GFP载体连接产生短发卡RNA慢病毒载体,用插入鉴定引物进行PCR鉴定阳性克隆并测序,应用Western blot在293T细胞中鉴定MMP-9表达的下调作用.结果 PCR鉴定和DNA测序结果显示合成的含MMP-9 shRNA慢病毒载体寡核苷酸链插入正确;构建的MMP-9RNAi慢病毒载体能够抑制MMP-9的表达.结论 应用基因工程技术,成功构建了小鼠MMP-9基因RNAi慢病毒载体,为应用于在体基因治疗创造了条件.
目的 構建小鼠基質金屬蛋白酶-9(matrix metalloproteI_(Na)se-9,MMP-9)RNA榦擾(RNA interference,RNAi)慢病毒載體.方法 針對小鼠MMP-9基因序列,利用公用網站按照RNAi序列設計原則,設計RNAi靶點序列,併閤成靶序列的Oligo DNA,退火形成雙鏈DNA,與經HpaⅠ和XhoⅠ酶切後的pGCL-GFP載體連接產生短髮卡RNA慢病毒載體,用插入鑒定引物進行PCR鑒定暘性剋隆併測序,應用Western blot在293T細胞中鑒定MMP-9錶達的下調作用.結果 PCR鑒定和DNA測序結果顯示閤成的含MMP-9 shRNA慢病毒載體寡覈苷痠鏈插入正確;構建的MMP-9RNAi慢病毒載體能夠抑製MMP-9的錶達.結論 應用基因工程技術,成功構建瞭小鼠MMP-9基因RNAi慢病毒載體,為應用于在體基因治療創造瞭條件.
목적 구건소서기질금속단백매-9(matrix metalloproteI_(Na)se-9,MMP-9)RNA간우(RNA interference,RNAi)만병독재체.방법 침대소서MMP-9기인서렬,이용공용망참안조RNAi서렬설계원칙,설계RNAi파점서렬,병합성파서렬적Oligo DNA,퇴화형성쌍련DNA,여경HpaⅠ화XhoⅠ매절후적pGCL-GFP재체련접산생단발잡RNA만병독재체,용삽입감정인물진행PCR감정양성극륭병측서,응용Western blot재293T세포중감정MMP-9표체적하조작용.결과 PCR감정화DNA측서결과현시합성적함MMP-9 shRNA만병독재체과핵감산련삽입정학;구건적MMP-9RNAi만병독재체능구억제MMP-9적표체.결론 응용기인공정기술,성공구건료소서MMP-9기인RNAi만병독재체,위응용우재체기인치료창조료조건.
Objective To construct lentiviral vector of matrix metalloproteI_(Na)se 9 (MMP-9) RNA interference (RNAi) of mice. Methods Towards the MMP-9 gene sequences of mice, in accordance with RNAi sequence design principles using the public Web site, RNAi target sequences were designed, then the target sequences of Oligo DNA were synthesized and annealed to double-stranded DNA, connecting with pGCL-GFP vector digested by Hpa Ⅰ and Xho Ⅰ. Short hairpin RNA lentiviral vectors were constructed. The positive clones identified by PCR and sequenced. The MMP-9 expression in the downward effect in 293T cells by Western blot was studied. Results The PCR identification and DNA sequencing showed that the lentiviral vector of MMP-9 shRNA-oligonucleotide chain was inserted correctly;and MMP-9 RNAi lentiviral vectors inhibited the expression of MMP-9. Conclusion A lentiviral vector of MMP-9 gene RNAi of mice is constructed successfully by the genetic engineering technology, and it provides a condition for gene therapy in vivo.
