lncRNA-uc.412重组质粒构建及在大鼠肾小球系膜细胞中的转染

Construction of lncRNA-uc.412 recombinant plasmid and its expression in rat mesangial cells

目的 构建长链非编码RNA-uc.412(lncRNA-uc.412)的重组质粒,并观察其转染后在大鼠肾小球系膜细胞中的表达情况。方法 自大鼠系膜细胞中提取总RNA,根据lncRNA-uc.412的基因信息设计上下游引物,应用PCR技术与琼脂糖凝胶电泳获得扩增后分离纯化的lncRNA-uc.412基因片段。将lncRNA-uc.412基因片段质粒与pRP[Exp]经双酶切处理后,通过凝胶电泳分离纯化,加入T4-DNA连接酶,即获得lncRNA-uc.412重组质粒。将lncRNA-uc.412重组质粒进行双酶切鉴定,检测目的基因片段大小;选取经双酶切鉴定连接成功的lncRNA-uc.412重组质粒,PCR扩增后进行双向测序。采用脂质体转染法转染lncRNA-uc.412重组质粒和空白质粒pRP[Exp]至大鼠肾小球系膜细胞,分别记为实验组和阴性对照组,采用RT-qPCR法检测lncRNA-uc.412在大鼠肾小球系膜细胞中的相对表达量。结果 lncRNA-uc.412重组质粒的双酶切鉴定结果显示,得到大小在200~300bp的特异性条带,与lncRNA-uc.412的长度相符。测序结果显示,lncRNA-uc.412重组质粒的碱基序列与理论预期序列一致。阴性对照组大鼠肾小球系膜细胞中lncRNA-uc.412的相对表达量记为1.00±0.00,实验组大鼠肾小球系膜细胞中lncRNA-uc.412的相对表达量为8.01±0.97,两组相比,P<0.05。结论 成功构建了lncRNA-uc.412重组质粒,并成功转染至大鼠肾小球系膜细胞。

Objective To construct the recombinant plasmid of long non-coding RNA-uc.412(lncRNA-uc.412) and to obverse its expression in rat mesangial cells after transfection. Methods Total RNA was extracted from rat mesangial cells. Upstream and downstream primers were designed according to the gene information of lncRNA-uc.412. The lncRNA-uc.412 gene fragment isolated and purified after amplification was obtained by PCR and agarose gel electrophoresis. The plasmid vector pRP[Exp] and lncRNA-uc.412 gene fragment were digested by same enzyme, and then were amplified by PCR and purified by agar gel electrophoresis, and then we obtained the recombinant plasmid after adding T4 ligase. The lncRNA-uc.412 recombinant plasmid was identified by restriction enzyme digestion and sequencing. Rat mesangial cells were transfected with the recombinant plasmid as the experimental group and with the blank plasmid as the control group by lipofection. The relative expression of lncRNA-uc.412 in the rat mesangial cells was detected by RT-qPCR. Results The double enzyme digestion indicated that the length of resulting band was consistent with the length of lncRNA-uc.412, which was about 200-300 bp. The base pairs of the lncRNA-uc.412 recombinant plasmid were correct by sequencing. The relative expression of the lncRNA-uc.412 in the control group was 1.00±0.00, and the relative expression in the experimental group was 8.01±0.97, with statistically significant difference ( P <0.05). Conclusion The lncRNA-uc.412 recombinant plasmid is constructed and transfected into rat mesangial cells successfully.