NELL2基因真核表达载体和microRNA干扰质粒的构建及其体外干预效应的研究

Construction of eukaryotic expression vector and microRNA expression plasmid of NELL2 gene and their biological effects in vitro

目的构建大鼠NELL2基因真核表达载体pcDNA3.1(-)-NELL2的microRNA(miRNA)干扰质粒,观察其转染体外细胞系后对NELL2 mRNA表达的干预效应。方法从SD大鼠下丘脑组织中提取总RNA,设计特异性引物,采用RT-PCR技术扩增NELL2基因全长cDNA,利用T载体将目的片段克隆至表达载体pcDNA3.1(-),得到重组质粒pcDNA3.1(-)-NELL2。针对NELL2基因分别设计4对pre-miRNA序列,通过T4连接酶克隆至pcDNA6.2-GW/EmGFP-miRNA表达载体构建干扰质粒(p-NELL2-miRNA1～4)。将p-NELL2-miRNA1～4分别与pcDNA3.1(-)-NELL2共转染人胚肾细胞系(HEK293)(p-NELL2-miRNA1～4干预组),采用Real-Time PCR技术检测转染后48 h细胞NELL2 mRNA表达;以转染pcDNA3.1(-)-NELL2细胞作为阳性对照组,以共转染pcDNA3.1(-)-NELL2和阴性对照干扰质粒pre-miRNA-neg细胞作为阴性对照组。结果酶切和测序鉴定结果表明:重组质粒pcDNA3.1(-)-NELL2和干扰质粒p-NELL2-miRNA的片段大小与预计相符,且克隆序列正确。Real-Time PCR结果显示:干预组细胞NELL2 mRNA表达显著低于阳性和阴性对照组(P<0.05或P<0.01)。结论成功构建NELL2基因真核表达载体及其microRNA干扰质粒,证实干扰质粒对HEK293细胞NELL2 mRNA表达具有特异性的抑制效应。

Objective To construct the microRNA(miRNA) expression plasmid(p-NELL2-miRNA) of eukaryotic expression vector of rat NELL2 gene(pcDNA3.1(-)-NELL2),and investigate its in vitro effect on expression of NELL2 mRNA. Methods Total RNA was extracted from hypothalamus of SD rats,specific primers were designed,cDNA of NELL2 gene was amplified by RT-PCR,and recombinant plasmid pcDNA3.1(-)-NELL2 was obtained after cloning of objective fragment to expression vector pcDNA3.1(-) with T vector.Four pairs of pre-miRNA sequences were designed for NELL2 gene,and were cloned to pcDNA6.2-GW/EmGFP-miRNA expression vector by T4 ligase for the construction of expression plasmid(p-NELL2-miRNA1-4).Human embryonic kidney 293 cells(HEK293) were co-transfected with p-NELL2-miRNA1-4 and pcDNA3.1(-)-NELL2(p-NELL2-miRNA1-4 treatment group),and the expression of NELL2 mRNA was detected by Real-Time PCR 48 h after transfection.Cells transfected with pcDNA3.1(-)-NELL2 were served as positive control group,and those co-transfected with pcDNA3.1(-)-NELL2 and negative control expression plasmid pre-miRNA-neg as negative control group. Results Enzyme digestion and sequencing revealed that the sizes of fragments of recombinant plasmid pcDNA3.1(-)-NELL2 and expression plasmid p-NELL2-miRNA were in line with those of anticipation,with correct cloned sequences.Real-Time PCR indicated that the expression of NELL2 mRNA in treatment group was significantly lower than that in positive control group and negative control group(P<0.05 or P<0.01). Conclusion The eukaryotic expression vector of NELL2 gene and microRNA expression plasmid have been successfully constructed,and microRNA expression plasmid has specific inhibitory effect on expression of NELL2 mRNA of HEK293 cells.