Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to C5aR gene
ZHANG Ze-ying1, 2, GAO Xiao-long2, LIU Dong-xu2*
Author information+
College of Food and Environmental Engineering, Wuchang Institute of technology, Wuhan 430065, China; 2.School of Life Sciences, Hubei University, Wuhan 430062, China
We aimed to construct a short hairpin RNA (shRNA) eukaryotic expression plasmids targeting C5aR gene, and to study the function of the protein C5aR and its role in the immunotherapy. According to the C5aR mRNA nucleotide sequence (NM_053619) provided in GENEBANK database, shRNA targeting the C5aR 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-52E through lipofectamine 2000. After treated with LPS, the knockdown efficiency of these shRNA expression plasmids on C5aR 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+C5aR shRNA group. Serum creatinine (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 C5aR shRNA, the expression level of C5aR mRNA and protein in NRK-52E cells decreased significantly (P < 0.05). In LPS induced acute kidney injury Wistar rats, the C5aR shRNA plasmid inhibited the LPS induced TNF-αand IL-6 expressions, and improved kidney functions. In conclusion, the recombinant interference plasmid could decrease C5aR gene expression effectively and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.
ZHANG Ze-ying, GAO Xiao-long, LIU Dong-xu.
Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to C5aR gene. Current Immunology. 2018, 38(3): 197-202