SPARC基因RNAi慢病毒载体的构建及其在SKM-1细胞中的表达

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。

Construction and identification of SPARC shRNA lentiviral vector and its expression in human SKM-1 cells

AIM: To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting SPARC gene and investigate its silenced effect on SPARC in human myelodysplastic syndromes(MDS) cell line SKM-1.METHODS: The targeting sequence of SPARC gene which can be effectively silenced in RNA interference was confirmed in our previous study.The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear pGCSIL-GFP lentiviral plasmid digested by enzyme Age I and EcoR I to produce GC-shSPARC lentiviraL vector.After being identified by PCR and sequencing,plasmids GC-shSPARC with pHelper 1.0 and pHelper 2.0 were cotransfected into 293T cells to package lentiviral particles.The recombinant lentiviral vector was transfected into human SKM-1 cells,transfection efficiency was evaluated with expression of green fluorescent protein(GFP) determined by fluorescent microscope.Expression of SPARC in SKM-1 cells was detected using RT-PCR and Western blotting.RESULTS: A recombinant lentiviral vector,GC-shSPARC,expressing shRNAs targeting SPARC gene was constructed and confirmed by DNA sequencing.The recombinant lentivirus was harvested from 293T cells with a viral titer of 1×109 TU/mL.GFP was observed in the 70% of SKM-1 cells after transfection.Expression of SPARC mRNA and protein was significantly reduced in the GC-shSPARC transfected group than that in the control group(P<0.05).CONCLUSION: The lentivirus RNAi vector targeting SPARC has been successfully constructed,and can effectively inhibit the expression of SPARC in SKM-1 cell line,which shed light on the foundation for researching the inhibition of SPARC siRNA target against human MDS cells proliferation,induction apoptosis and gene therapy.