Construction Of Human Interleukin-10 Eukaryotic Expression Vector PcDNA4/HismaxA-hIL-10 And Its Expression In Rabbit Synovial Cells

OBJECTIVETo clone human interleukin-10 (hIL-10) full-length open reading frame(ORFs) from human peripheral blood mononuclear cells(PBMC),and to construct an efficient eukaryotic expression recombinant vector of hIL-10, and to observe its expression in rabbit synovial cells(RSCs), then to evaluate the possibility of gene therapy for rheumatoid arthritis by using hIL-10.METHODSTotal RNA was extracted from PBMC of a patient with drug allergy. With total RNA as a template, cDNA was synthesized by Oligo(dT) primer. Specific primers for full-length ORFs of hIL-10 were designed according to GeneBank (NM 000572). Full-length ORFs of hIL-10 were amplified by reverse thanscription polymerase reaction (RT-PCR). PCR products (≈0.54kb) were digested by restrictive endonucleotidase, then inserted into plasmid pcDNA4/HisMaxA for recombinants construction. Both endonucleotidase analysis and DNA sequencing were carried out for inserts verification. RSCs were transfected with recombinant plasmid expressing vector pcDNA4/HisMaxA-hIL-10 byliposome-mediated gene transfer method , then cultured in vitro. The supernatants were collected after transfection in 12h, 24h, 48h, 72h, 7d, 14d respectively for IL-10 measurement by enzyme linked immunosorbent assay(ELISA). The SPSS (Verson...