大鼠nelin基因干扰质粒的构建及其体外抑制效果研究

The Construction and in vitro Study of Inhibition Effect of shRNA Plasmid targeting rat nelin

目的构建大鼠nelin基因干扰质粒并对其进行体外抑制效果验证,为研究nelin在心脏发育、心肌肥厚、心衰及其它心脏疾病中的作用提供有效的研究工具。方法提取大鼠心肌组织RNA,逆转录为cDNA,设计合成含BamHI和EcoRI酶切位点的引物,以大鼠cDNA为模板PCR扩增获得nelin基因,定向克隆入pEGFP-N1-3FLAG载体,测序验证。以克隆得到的大鼠nelin基因为模板,设计合成候选干扰序列,亚克隆至pGCSIL-U6载体,获得干扰质粒。以nelin基因干扰质粒和重组表达质粒共转染HEK293T细胞,WesternBlot方法检测干扰效果。结果成功克隆了大鼠nelin基因并构建了nelin基因重组表达载体pEGFP-N1-3FLAG-rNelin。设计构建的4个干扰质粒(靶位点分别为+370-388位,+376-394位,+545-563位,+1206-1224,命名为pGCSIL-U6-nelin/siteⅠ,Ⅱ,Ⅲ,Ⅳ)均可显著降低nelin基因的表达,尤以靶向(+370-388位)位点即pGCSIL-U6/nelin-siteI质粒的干扰效果最佳。结论本实验构建的大鼠nelin基因特异性干扰质粒能从蛋白水平上高效特异地抑制ne-lin基因表达,为进一步开展其功能研究奠定了基础。

Objective To provide a useful tool for studying the function of nelin during the heart development,hypertrophy,heart failure and other heart diseases,the RNA-interference plasmids targeting rat nelin gene were constructed,of which silencing effect were evaluated in this study.Methods A pair of primers based on human nelin gene with BamH I and EcoR I restriction site were designed and synthesized.Fragment was amplified with cDNA template reversed from rat heart RNA and cloned into the pEGFP-N1-3FLAG vector,which was identified by sequencing.Four specific interference sequences targeting rat nelin （the target sites are ＋370-388,＋376-394,＋545-563 and ＋1206-1224） were designed,synthesized and inserted into the pGCSIL-U6 vector respectively.RNA interference plasmids （pGCSIL-U6-nelin/site Ⅰ,Ⅱ,Ⅲ,Ⅳ） and recombinant expression plasmids （pEGFP-N1-3FLAG-rNelin） were co-transfected into HEK293T cells.The expression of Nelin-FLAG was detected by western blot.Results pEGFP-N1-3FLAG-rNelin was constructed successfully.All of the RNA interference plasmids constructed in this study could reduce the expression of rat Nelin in 293T cell line,especially the pGCSIL-U6-nelin/site I had the ideal distinct effect.Conclusion Rat nelin-specific RNA interference plasmids constructed in this research,as an effective tool for further function study of Nelin,can knock down the expression of rat nelin efficiently in protein level.