摘要
为了构建绵羊PLC-γ1基因shRNA慢病毒干扰载体,并在人胚肾细胞(HEK-293T细胞)中筛选及鉴定载体干扰效果,试验采用Invitrogen BLOCK-iTTM RNAi Designer在线软件设计PLC-γ1基因的4条shRNA干扰序列(PLC-γ1-shRNA-1~4)和1条阴性对照序列(PLC-γ1-shRNA-NC),将其分别克隆至慢病毒干扰载体pLentiLox3.7(pLL3.7)中构建PLC-γ1-shRNA慢病毒干扰载体,用其转染HEK-293T细胞,48 h后通过倒置荧光显微镜观察荧光蛋白表达情况,并利用实时荧光定量PCR法和Western-blot检测HEK-293T细胞中PLC-γ1基因和蛋白质的表达情况,筛选出干扰效率最高的PLC-γ1-shRNA干扰序列并计算慢病毒含量。结果表明:试验成功设计出PLC-γ1-shRNA慢病毒干扰载体,大小为120 bp。PLC-γ1-shRNA慢病毒干扰载体均具有较高的干扰效率,在倒置荧光显微镜下均可观察到绿色荧光蛋白表达,其中PLC-γ1-shRNA-2序列的干扰效率最高,其PLC-γ1基因和蛋白质的相对表达量最低,极显著低于PLC-γ1-shRNA-NC的干扰(P<0.01),慢病毒含量为1.1×10^(7)IU/mL。说明针对PLC-γ1基因筛选出的PLC-γ1-shRNA慢病毒干扰载体从基因和蛋白质水平上均能有效地抑制PLC-γ1的表达。
In order to construct a shRNA lentiviral interference vector of sheep PLC-γ1 gene and to screen and identify the interference effect of the vector in human embryonic kidney cells(HEK-293T cells),in the experiment,Invitrogen BLOCK-iTTM RNAi Designer software was used to design four shRNA interference sequences(PLC-γ1-shRNA-1 to 4)and one negative control(PLC-γ1-shRNA-NC)of PLC-γ1 gene,which were cloned into pLentiLox3.7(pLL3.7)plasmid respectively to construct the PLC-γ1 lentiviral interference vectors.HEK-293T cells were transfected with them,and the fluorescence expression was observed by fluorescence microscope after 48 h.The expression of PLC-γ1 gene and protein in HEK-293T cells was detected by real-time fluorescence quantification and Western-blot;shRNA lentiviral interference vectors with the highest interference efficiency were screened out and their lentiviral contents were calculated.The results showed that the experiment successfully designed shRNA lentiviral interference vectors with a size of 120 bp.shRNA lentiviral interference vectors all had high interference efficiency,and green fluorescent protein expression could be observed under the inverted fluorescence microscope in all of them.The interference efficiency of PLC-γ1-shRNA-2 was the highest,and the relative expression of PLC-γ1 gene and protein was the lowest,which was highly significantly lower than that of PLC-γ1-shRNA-NC(P<0.01);its lentiviral content was 1.1×107 IU/mL.The results suggested that the shRNA interference vector screened against PLC-γ1 gene could effectively inhibit the expression of PLC-γ1 at both gene and protein levels.
作者
陈云蕾
袁利明
丁林玲
刘素平
袁伟琴
赛务加甫
CHEN Yunlei;YUAN Liming;DING Linling;LIU Suping;YUAN Weiqin;Saiwujiafu(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Tonglu County Management Center for Unregulated Equine Disease Zones,Hangzhou 311599,China;College of Veterinary Medicine,Northwest Agriculture and Forestry University,Yangling 712100,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2024年第13期52-58,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31860725)。
