医学研究生学报
醫學研究生學報
의학연구생학보
JOURNAL OF MEDICAL POSTGRADUATE
2015年
2期
136-140
,共5页
马卫列%丁航%李观强%肖娟%张志珍
馬衛列%丁航%李觀彊%肖娟%張誌珍
마위렬%정항%리관강%초연%장지진
人β-COP%shRNA干扰载体%脂质代谢
人β-COP%shRNA榦擾載體%脂質代謝
인β-COP%shRNA간우재체%지질대사
Human β-COP%Short hairpin RNA interference vector%Lipid metabolism
目的:采用常规转染试剂难以将人β-COP-shRNA转染至THP-1细胞。构建人β-COP特异性的shRNA慢病毒载体,并测定其对靶基因β-COP的抑制效应。方法设计合成针对人β-COP基因靶点特异的4对shRNA寡聚单链DNA,插入pGMLV-SC1载体中,构建慢病毒重组载体。将重组载体和包装质粒共转染HEK 293 T细胞,包装产生慢病毒并测定滴度。用慢病毒感染THP-1细胞,定量PCR 和Western blot 检测干扰载体对β-COP基因的干扰效果。结果测序证实,β-COP基因的shRNA寡聚核苷酸序列已插入慢病毒载体,转染HEK 293T细胞,经包装得到4种慢病毒,病毒滴度为1×109 TU/mL。定量PCR分析显示,4种β-COP-shRNA 慢病毒感染的 THP-1细胞中β-COP基因 mRNA 表达量分别为0.831±0.065、0.675±0.079、0.260±0.050、0.497±0.067,较对照(1.000±0.000)明显升高( P<0.01);其抑制率分别为16.9%、32.5%、74.0%和50.3%。 Western blot结果表明,4种干扰载体均可抑制β-COP蛋白表达,其中β-COP-shRNA 3的沉默效率为76.4%。结论成功构建了人β-COP-shRNA干扰载体,证实了干扰载体对靶基因有较好的沉默效果。
目的:採用常規轉染試劑難以將人β-COP-shRNA轉染至THP-1細胞。構建人β-COP特異性的shRNA慢病毒載體,併測定其對靶基因β-COP的抑製效應。方法設計閤成針對人β-COP基因靶點特異的4對shRNA寡聚單鏈DNA,插入pGMLV-SC1載體中,構建慢病毒重組載體。將重組載體和包裝質粒共轉染HEK 293 T細胞,包裝產生慢病毒併測定滴度。用慢病毒感染THP-1細胞,定量PCR 和Western blot 檢測榦擾載體對β-COP基因的榦擾效果。結果測序證實,β-COP基因的shRNA寡聚覈苷痠序列已插入慢病毒載體,轉染HEK 293T細胞,經包裝得到4種慢病毒,病毒滴度為1×109 TU/mL。定量PCR分析顯示,4種β-COP-shRNA 慢病毒感染的 THP-1細胞中β-COP基因 mRNA 錶達量分彆為0.831±0.065、0.675±0.079、0.260±0.050、0.497±0.067,較對照(1.000±0.000)明顯升高( P<0.01);其抑製率分彆為16.9%、32.5%、74.0%和50.3%。 Western blot結果錶明,4種榦擾載體均可抑製β-COP蛋白錶達,其中β-COP-shRNA 3的沉默效率為76.4%。結論成功構建瞭人β-COP-shRNA榦擾載體,證實瞭榦擾載體對靶基因有較好的沉默效果。
목적:채용상규전염시제난이장인β-COP-shRNA전염지THP-1세포。구건인β-COP특이성적shRNA만병독재체,병측정기대파기인β-COP적억제효응。방법설계합성침대인β-COP기인파점특이적4대shRNA과취단련DNA,삽입pGMLV-SC1재체중,구건만병독중조재체。장중조재체화포장질립공전염HEK 293 T세포,포장산생만병독병측정적도。용만병독감염THP-1세포,정량PCR 화Western blot 검측간우재체대β-COP기인적간우효과。결과측서증실,β-COP기인적shRNA과취핵감산서렬이삽입만병독재체,전염HEK 293T세포,경포장득도4충만병독,병독적도위1×109 TU/mL。정량PCR분석현시,4충β-COP-shRNA 만병독감염적 THP-1세포중β-COP기인 mRNA 표체량분별위0.831±0.065、0.675±0.079、0.260±0.050、0.497±0.067,교대조(1.000±0.000)명현승고( P<0.01);기억제솔분별위16.9%、32.5%、74.0%화50.3%。 Western blot결과표명,4충간우재체균가억제β-COP단백표체,기중β-COP-shRNA 3적침묵효솔위76.4%。결론성공구건료인β-COP-shRNA간우재체,증실료간우재체대파기인유교호적침묵효과。
[Abstract ] Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the humanβ-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized 4 humanβ-COP-specific oligonucleotide sequences and inserted them into the pGMLV-SC1 vector to construct a recombinant vector fol-lowed by transfection of HEK 293T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral con-tent.After infecting the THP-1 cells with the packaged lentiviruses , we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot . Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were in-serted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293T cells, with the final viral content of 1 × 109 TU/mL.Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP (P<0.01), with interference rates of 16.9 %,32.5%, 74.0%, and 50.3%, respectively.Western blot also indicated their inhibitory effect on the protein expression of β-COP, with an inhibition rate of 76.4% onβ-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully , which could suppress the expression of the human β-COP gene.