Abstract:

Objective To construct the shRNA interference vector and over-expression vector of N-myc downstream regulatory gene 1 （NDRG1） and transfect the human cerebral microvascular endothelial cells （hCMECs），and to verify the interference and over-expression effects and to obtain the cells stably over-expressing NDRG1. Methods The pGPU6-GFP plasmid was double digested by BamH Ⅰ and Bbs Ⅰ， and then ligated with eight designed shRNAs to construct the pGPU6-GFP-NDRG1 shRNA interference vectors respectively，and the hCMECs were transfected by Lipofectamine 2000 after sequencing and identification， and the transfection efficiency of the vectors was observed under fluorescence microscope； the sequence of NDRG1 coding region amplified by PCR was ligated with the pMD19-T， after correct sequencing， the recombinant plasmid and the empty plasmid pcDNA3.1 were digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ， and the pcDNA3.1-NDRG1 over-expression vector was constructed after ligation；the hCMECs were transfected with the correctly sequenced over-expression vector by using Lipofectamine 2000；the expression levels of NDRG1 mRNA in the hCMECs were detected by real-time fluorescence quantitative PCR （RT-qPCR） method，and the expression levels of NDRG1 protein in the hCMECs were detected by Western blotting method；the positive clone hCMECs were screened by neomycin. Results After the pGPU6-GFP vector was doubly digested，the electrophoresis results showed that it was completely linear， and the sequencing results showed that the shRNAs were all successfully connected to the pGPU6-GFP vector；the transfection efficiency of vector was about 70 % observed under fluorescence microscope； the RT-qPCR results showed that there were three shRNA interference vectors with good interference effects， and the expression levels of NDRG1 mRNA in the hCMECs after transfection were about 26%， 21%， and 13% of those in control group， respectively；the corresponding specific protein bands were obtained by Western blotting method，the recombinant plasmid pcDNA3.1-NDRG1 was identified by enzymatic digestion， and two DNA bands of 1 185 and 5 428 bp were identified by electrophoresis，and the sequencing results showed that the NDRG1 gene was successfully inserted into the pcDNA3.1 vector， and the positive clone hCMECs were successfully constructed.The RT-qPCR results showed that the expression level of NDRG1 in the positive clone hCMECs was about 25.96 times higher than that in control group； the Western blotting results showed that the expression level of NDRG1 protein in the hCMECs was increased compared with control group. Conclusion The pGPU6-GFP-NDRG1 shRNA interference vector is successfully constructed and the interference effect is verified； the pcDNA3.1-NDRG1 over-expression vector and the hCMECs stably over-expressing NDRG1 are successfully constructed.

Key words: N-myc downstream regulated gene 1, RNA interference, Eukaryotic expression vector, Over expression, Human brain microvascular endothelial cells