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目的:构建SPARC基因RNAi慢病毒载体获得稳定产毒的细胞,并观察其对人MDS细胞株SKM-1细胞的转染效率及其对SPARC基因的抑制效率。方法:针对已经筛选确定的SPARC基因RNAi有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生GC-shSPARC慢病毒载体,PCR筛选阳性克隆并进行测序鉴定。用GC-shSPARC、pHelper1.0和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度,并将获得的重组慢病毒GC-shSPARC转染SKM-1细胞,通过荧光显微镜检测转染后GFP表达情况,测定转染效率;RT-PCR和Western blot分别验证转染后SKM-1细胞SPARC mRNA及蛋白的表达。结果:经测序证实,构建出了SPARC shRNA的慢病毒载体GC-sh SPARC。包装、浓缩病毒悬液的滴度为1×109TU/mL。荧光显微镜下能直接观察到转染组细胞的GFP表达,转染效率为70%,RT-PCR、Western blot技术分别检测到GC-shSPARC慢病毒转染SKM-1细胞SPARC mRNA、SPARC蛋白表达水平较空白组明显降低(P<0.05)。结论:成功构建SPARC基因RNAi慢病毒载体,其能高效干扰SKM-1细胞SPARC基因的表达。
OBJECTIVE: To construct stably genetically engineered cells expressing the RNAi lentiviral vector of SPARC gene and to observe its transfection efficiency and its inhibitory effect on SPARC gene expression in human MDS cell line SKM-1. Methods: According to the RNAi effective target sequences of the SPARC gene that have been screened, the target sequence OligoDNA was synthesized and annealed to form the double-stranded DNA. The double-stranded DNA was ligated with the pGCSIL-GFP vector digested with AgeⅠ and EcoRⅠ to generate the GC-shSPARC lentiviral vector. PCR Positive clones were screened and sequenced. The 293T cells were co-transfected with 293T cells with GC-shSPARC, pHelper1.0 and pHelper2.0 vectors, and then lentivirus was packaged to determine the virus titer with the expression level of GFP protein of 293T cells. The recombinant lentivirus GC-shSPARC The expression of GFP in SKM-1 cells was detected by fluorescence microscopy. The transfection efficiency was determined. The expression of SPARC mRNA and protein in SKM-1 cells was verified by RT-PCR and Western blot respectively. Results: After sequencing confirmed that the SPARC shRNA lentiviral vector GC-sh SPARC was constructed. Packaging, concentrated virus suspension titer 1 × 109TU / mL. The GFP expression in the transfection group was observed under a fluorescence microscope. The transfection efficiency was 70%. The SPARC mRNA and SPARC protein levels were detected by RT-PCR and Western blot in the SK-1 cells transfected with GC-shSPARC lentivirus Compared with the blank group was significantly lower (P <0.05). CONCLUSION: The RNAi lentiviral vector of SPARC gene is successfully constructed, which can effectively interfere the expression of SPARC gene in SKM-1 cells.