NSPc1-siRNA重組慢病毒的構(gòu)建及其對U87細(xì)胞NSPc1基因的沉默效應(yīng)
發(fā)布時(shí)間：2019-02-11 16:22
【摘要】：【目的】構(gòu)建包裝NSPc1-si RNA慢病毒顆粒,并在U87膠質(zhì)瘤細(xì)胞中鑒定其感染及基因沉默效果�！痉椒ā扛鶕�(jù)Gen Bank中基因信息,采用干擾序列設(shè)計(jì)軟件設(shè)計(jì)靶點(diǎn),制備合成GV118-si RNA目的質(zhì)粒,轉(zhuǎn)化感受態(tài)細(xì)胞,對于長出的克隆應(yīng)用菌落PCR鑒定,再對PCR鑒定陽性的克隆進(jìn)行測序和對比分析,重組病毒質(zhì)粒與另外2種輔助包裝載體質(zhì)粒通過Lipofectamine TM2 000共轉(zhuǎn)染293T細(xì)胞,培養(yǎng)48 h后,收集細(xì)胞培養(yǎng)上清液,將病毒濃縮后在293T細(xì)胞中測定病毒滴度;并檢測病毒顆粒在目的細(xì)胞U87膠質(zhì)瘤細(xì)胞中的感染效率,實(shí)時(shí)熒光定量PCR(RT-PCR)和Western blotting方法檢測NSPc1 si RNA慢病毒對U87中NSPc1的干擾作用�！窘Y(jié)果】成功構(gòu)建NSPc1 si RNA慢病毒載體GV118-si RNA,重組病毒滴度為4×108TU/ml;用該病毒體外感染U87膠質(zhì)瘤細(xì)胞,當(dāng)感染復(fù)數(shù)(MOI)為10時(shí),感染效率大于90%;RT-PCR和Western blotting方法檢測NSPc1基因沉默的效率分別為66.4%、60.0%�！窘Y(jié)論】成功構(gòu)建了NSPc1 si RNA慢病毒載體GV118-si RNA,該重組病毒包裝后在體外感染U87膠質(zhì)瘤細(xì)胞的效率較高,且具有顯著的基因沉默效果。
[Abstract]:[objective] to construct the packaged NSPc1-si RNA lentivirus particles and identify the effect of infection and gene silencing in U87 glioma cells. [methods] according to the gene information in Gen Bank, the target was designed by interference sequence design software. The target plasmid of GV118-si RNA was prepared and transformed into receptive cells. The clones were identified by colony PCR, and the positive clones identified by PCR were sequenced and compared. The recombinant virus plasmid and the other two kinds of auxiliary package vector plasmids were co-transfected into 293T cells by Lipofectamine TM2 000. After 48 hours of culture, the supernatants of cell culture were collected and the virus titers were measured in 293T cells after the virus was concentrated. The infection efficiency of virus particles in U87 glioma cells was detected. Real-time fluorescence quantitative PCR (RT-PCR) and Western blotting were used to detect the interference of NSPc1 si RNA lentivirus to NSPc1 in U87. [results] the recombinant NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed with a titer of 4 脳 108 TU / ml. U87 glioma cells were infected with the virus in vitro. When the complex (MOI) was 10:00, the infection efficiency was greater than 90%. The detection efficiency of NSPc1 gene silencing by RT-PCR and Western blotting was 66.4% and 60.0% respectively. [conclusion] NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed. The recombinant virus can infect U87 glioma cells in vitro with high efficiency and remarkable gene silencing effect.
【作者單位】： 武警后勤學(xué)院生物化學(xué)與分子生物學(xué)教研室;武警后勤學(xué)院人體解剖與組織胚胎學(xué)教研室;武警后勤學(xué)院附屬醫(yī)院腦科醫(yī)院神經(jīng)內(nèi)科;
【基金】：國家自然科學(xué)基金青年項(xiàng)目(81201757) 天津市自然科學(xué)基金青年項(xiàng)目(13JCQNJC09600) 武警后勤部項(xiàng)目(WJHQ2012-13)
【分類號】：R346
[Abstract]:[objective] to construct the packaged NSPc1-si RNA lentivirus particles and identify the effect of infection and gene silencing in U87 glioma cells. [methods] according to the gene information in Gen Bank, the target was designed by interference sequence design software. The target plasmid of GV118-si RNA was prepared and transformed into receptive cells. The clones were identified by colony PCR, and the positive clones identified by PCR were sequenced and compared. The recombinant virus plasmid and the other two kinds of auxiliary package vector plasmids were co-transfected into 293T cells by Lipofectamine TM2 000. After 48 hours of culture, the supernatants of cell culture were collected and the virus titers were measured in 293T cells after the virus was concentrated. The infection efficiency of virus particles in U87 glioma cells was detected. Real-time fluorescence quantitative PCR (RT-PCR) and Western blotting were used to detect the interference of NSPc1 si RNA lentivirus to NSPc1 in U87. [results] the recombinant NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed with a titer of 4 脳 108 TU / ml. U87 glioma cells were infected with the virus in vitro. When the complex (MOI) was 10:00, the infection efficiency was greater than 90%. The detection efficiency of NSPc1 gene silencing by RT-PCR and Western blotting was 66.4% and 60.0% respectively. [conclusion] NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed. The recombinant virus can infect U87 glioma cells in vitro with high efficiency and remarkable gene silencing effect.
【作者單位】： 武警后勤學(xué)院生物化學(xué)與分子生物學(xué)教研室;武警后勤學(xué)院人體解剖與組織胚胎學(xué)教研室;武警后勤學(xué)院附屬醫(yī)院腦科醫(yī)院神經(jīng)內(nèi)科;
【基金】：國家自然科學(xué)基金青年項(xiàng)目(81201757) 天津市自然科學(xué)基金青年項(xiàng)目(13JCQNJC09600) 武警后勤部項(xiàng)目(WJHQ2012-13)
【分類號】：R346
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