Construction of a C3aR expres sion vector and infection in tubular epithelial cells / 医学研究生学报

ABSTRACT

Objective Our previous study showed that the expression of C 3aR was increased in renal tubular epithelial cells (RTEC).However, the role of C3aR in diabetic nephropathy remains unknown, and so does the exact physiological and pathological signifi-cance of C3aR in relevant renal tissues.In the present study, we investigated the physiological and pathological significance of C3aR in RTECs using a lentivirus expression vector and anRTEC strain overexpressing C3aR. Methods Based on the sequence of C3aR mRNA, the human C3aR gene was synthesized and cloned into the multi-clonal site of the lentivirus expression vector pLenti6.3-MCS-IRES2-EGFP to construct a C3aR expression vector pLenti6.3-C3aR-IRES2-EGFP.After identification by sequencing, the recombinant lentivirus expressing C3aR was packaged by cotransfecting293T cellswith the recombinant lentivirusexpression vector and packaged plasmid .Then,the recombinant lentivirus was used to infect the human RTECline HK 2.After screening in the medium with blasticidin, blasticidinr-esistant cell clones were obtained, followed by identification ofthe human RTECline stably overexpressing C 3aR by real-time PCR and immunochemical staining. Results TheC3aR expression vector pLenti6.3-C3aR-IRES2-EGFP was successfully constructed and the sequence was proved to be correct. C3aR expression recombinant lentivirus was successfully packaged with a titer of 5×108/mL.Blasticidin-resistant HK2 cell clones were ob-tained and the expression of HK2-C3aR mRNA was significantly higherin the HK2-C3aR cells than in the non-transfected HK2 cells (2.33± 0.45 sv 1.00±0.09, P<0.05). Conclusion We successfully constructed a C3aR expression lentivirus vector pLenti6.3-C3aR-IRES 2-EGFP and a C3aR overexpression renal tubular epithelial cell strain HK2-C3aR, which is very contributive to further studies of the roles of C3aR in renal tubular epithelial cells and other types of cells.