Abstract:

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.

Key words: NELL2, microRNA, eukaryotic expression vector, human embryonic kidney 293 cells