家兔DEPTOR基因过表达和shRNA干扰慢病毒载体的构建与验证  

Construction and validation of the overexpression and shRNA interfering lentiviral vectors of rabbit DEPTOR gene

作　　者：邹灵秀[1] 农恬颖 吴咏梅 邓彦飞[1] ZOU Lingxiu;NONG Tianying;WU Yongmei;DENG Yanfei(College of Animal Science and Technology,Guangxi Univesity,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《黑龙江畜牧兽医》2022年第23期7-13,131,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31760334);广西教育厅科研项目(2019KY0048)。

摘　　要：为了构建DEPTOR基因的过表达载体和shRNA干扰慢病毒载体,并验证载体的有效性,试验采用RT-PCR方法扩增兔DEPTOR基因编码区(CDS)序列,得到DEPTOR基因片段,同时合成DEPTOR基因的shRNA干扰序列。将DEPTOR基因片段分别连接到慢病毒真核表达载体pLVX-IRES-ZsGreen1和融合表达载体pEGFP-N1上,获得过表达载体pLVX-DEPTOR-IRES-ZsGreen1和pEGFP-DEPTOR;将shRNA干扰序列连接到慢病毒干扰载体pSicoR-Ef1a-mCh上,获得shRNA干扰慢病毒载体pSicoR-shRNA-508。用过表达载体转染HEK 293T细胞,72 h后观察荧光蛋白的表达情况并收集细胞,RT-PCR检测细胞水平上DEPTOR基因的表达情况。使用多质粒共同转染法,将过表达载体pEGFP-DEPTOR与shRNA干扰慢病毒载体pSicoR-shRNA-508按照1∶1比例转染HEK 293T细胞,72 h后观察荧光蛋白的表达情况并收集细胞,采用实时荧光定量PCR方法检测shRNA干扰序列对DEPTOR基因表达的干扰效率。结果表明:用构建的过表达载体pEGFP-DEPTOR和pLVX-DEPTOR-IRES-ZsGreen1转染HEK 293T细胞后均能观察到绿色荧光蛋白的表达,RT-PCR能扩增出相应条带,即慢病毒感染法能成功将DEPTOR基因转入细胞内。构建的shRNA干扰慢病毒载体pSicoR-shRNA-508能有效降低DEPTOR基因的表达,与未干扰相比差异显著(P<0.05),干扰效率约为80%。说明试验成功构建出DEPTOR基因的过表达载体和shRNA干扰慢病毒载体。In order to construct the overexpression recombinant lentivirus vector and shRNA interference lentiviral vector of the DEPTOR gene, the CDS sequence of rabbit DEPTOR gene was amplified by RT-PCR, and the fragment of DEPTOR gene was designed and amplified. Meanwhile, the shRNA interference sequence of DEPTOR gene was synthesized. The DEPTOR gene fragment was ligated to the lentiviral eukaryotic expression vector pLVX-IRES-ZsGreen1 and the fusion expression vector pEGFP-N1, respectively, to obtain the overexpression vectors pLVX-DEPTOR-IRES-ZsGreen1 and pEGFP-DEPTOR. The shRNA interference sequence was ligated to lentiviral interference vector pSicoR-Ef1 a-mCh to obtain the shRNA interference lentiviral vector pSicoR-shRNA-508. HEK 293 T cells were transfected with overexpression vectors. After 72 h, the expression of fluorescent protein was observed and the cells were collected. The expression of DEPTOR gene at the cell level was detected by RT-PCR. The recombinant plasmid pEGFP-DEPTOR and pSicoR-shRNA-508 were co-transfected into HEK 293 T cells at 1∶1 ratio by multi-plasmid co-transfection method. After 72 hours, the expression of fluorescent protein was observed and the cells were collected. The interference efficiency of shRNA on DEPTOR gene expression was detected by real-time PCR. The results showed that the expression of green fluorescent protein could be observed after transfection of HEK 293 T cells with the constructed overexpression vectors pEGFP-DEPTOR and pLVX-DEPTOR-IRES-ZsGreen1, and the corresponding bands could be amplified by RT-PCR, indicating that DEPTOR gene could be successfully transferred into cells by lentiviral infection. The constructed lentiviral vector pSicoR-shRNA-508 could effectively reduce the expression of DEPTOR gene, which was significantly different from the ones without interference(P<0.05), and the interference efficiency was about 80%. It indicated that the overexpression recombinant lentivirus vector and shRNA interference lentivirus vector of DEPTOR gene were successfull

关 键 词：DEPTOR基因 家兔 慢病毒载体 RNA干扰 转染 

分 类 号：S829.1[农业科学—畜牧学]