靶向大鼠C5aR基因shRNA真核表达载体的构建与鉴定

Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to C5aR gene

构建靶向大鼠C5aR基因的短发夹RNA(shRNA)表达载体,验证C5aR蛋白的功能,为后续进一步研究C5aR在免疫治疗中的作用奠定基础。具体方法是根据基因库提供的大鼠C5aR mRNA核苷酸序列(序列号:NM_053619),设计并合成短发夹结构的互补DNA序列,克隆到经BamHI和EcoR I双酶切的线性化质粒pLVX-shRNA,构建重组质粒,并予以DNA测序鉴定。重组干扰质粒构建成功后,通过脂质体2000转染大鼠肾小管上皮细胞NRK-52E,LPS处理后收集细胞,采用荧光定量RT-PCR(RT-qPCR)和Western blotting法检测干扰质粒对C5aR基因的表达抑制效应。通过尾静脉高压注射技术将重组干扰质粒导入Wistar大鼠肾脏,采用腹腔注射LPS方法复制脓毒血症急性肾损伤模型,大鼠随机分为正常对照组、LPS处理组、LPS+C5aR shRNA转染组,采用ELISA方法检测血清中Cr、BUN以及TNF-α、IL-6等炎性因子的含量。结果显示构建的shRNA序列经DNA测序证实与设计序列完全一致,重组质粒构建成功。转染C5aR shRNA后,NRK-52E细胞中C5aR mRNA和C5aR蛋白表达水平显著下降(P<0.05)。该重组干扰质粒能有效抑制C5aR基因的表达,能显著抑制由LPS诱导的TNF-α、IL-6等炎性因子的释放,改善大鼠肾脏功能,对LPS诱导的肾脓毒血症损伤具有明显保护作用。

We aimed to construct a short hairpin RNA（shRNA）eukaryotic expression plasmids targeting C5 aR gene,and to study the function of the protein C5 aR and its role in the immunotherapy.According to the C5 aR mRNA nucleotide sequence（NM_053619）provided in GENEBANK database,shRNA targeting the C5 aR gene were designed and synthesized,and then cloned into the pLVX-shRNA vectors which were digested by restriction endonucleases BamHI and EcoR I to construct recombinant plasmids.After identification by DNA sequencing,the shRNA expression vectors were transfected into rat renal tubular epithelial cell lines NRK-52 Ethrough lipofectamine 2000.After treated with LPS,the knockdown efficiency of these shRNA expression plasmids on C5 aR expression was evaluated by real time fluorescence based quantitative PCR（RT-qPCR）and Western blotting.Recombinant plasmids were imported into kidney of rats using tail vein high-pressure injection technology,then LPS was injected intraperitoneally into rats to construct sepsis acute kidney injury model.Wistar rats were randomly divided into normal control group,LPS group and LPS＋C5 aRshRNA group.Serum creatinin（Cr）,blood urea nitrogen（BUN）,TNF-αand IL-6 were detected by ELISA.The results showed that the recombinant shRNA sequence was confirmed by DNA sequencing,indicating the recombinant plasmid was constructed successfully.After transfection of C5 aRshRNA,the expression level of C5 aR mRNA and protein in NRK-52 Ecells decreased significantly（P〈0.05）.In LPS induced acute kidney injury Wistar rats,the C5 aRshRNA plasmid inhibited the LPS induced TNF-αand IL-6 expressions,and improved kidney functions.In conclusion,the recombinant interference plasmid could decrease C5 aR gene expression effectively and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.