Construction of human interleukin-10 eukaryotic expression vector pcDNA4/HisMaxA-hIL-10 and its expression in rabbit synovial cells
- VernacularTitle:人白细胞介素-10真核表达载体的构建及其在兔滑膜细胞中的表达
- Author:
Jibo WANG
;
Zhenhua Lü
;
Guoping LIU
;
Xiangping LIU
;
Yanming WANG
;
Kun YANG
;
Guangjie YU
;
Hongda LIANG
- Publication Type:Journal Article
- Keywords:
Interleukin-10;
Eukaryotic expression vector;
Transfection;
Rabbits:Synovial mem-brane
- From:
Chinese Journal of Rheumatology
2008;12(4):250-253
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an efficient eukaryotic expression recombinant vector of human interleukin-1O(hIL-lO),and observe its expression in rabbit synoviocytes(RSCs).Methods Total RNA was extracted from peripheral blood mononuclear cells(PBMCs)of a patient with drug allergy.Specific Drimers for full-length open reading frames(ORFs)of hIL-10 were designed according to GeneBank(NM 000572).Withtotal RNA as the template,full-length ORFs of hIL-10 were amplified by reverse transcription Dolymerase chain reaction(RT-PCR).RT-PCR products were digested by restrictive endonucleotidase.then inserted into plasmid pcDNA4/HisMaxA.Both restrictive endonucleotidase analysis and DNA sequencing Were carried out for inserts verification.RSCs were transfected with recombinant plasmid expression vector PcDNA4 HisMaxA-hiL10 by liposome-mediated gene transfer methods,then cultured in vitro.The supernatants were collected af-ter transfection for 12 hours,24 hours,48 hours,72 hours,7 days,14 days respectively for IL-10 measure-ment by enzyme linked immunosorbent assay(ELISA).Results Full-length ORFs of hIL-1o(0.54 kb)had been successfully cloned from PBMCs through RT-PCR.The inserts and insert location of pcDNA4 HisMaxA were in a fight way verified by enzyme analysis and DNA sequencing.ELISA results showed that exogenous hIL-10 gene had expressed in the transfected RSCs from 12 hours to 7 days after transfection,and hIL-10level of transfection group significantly higher than that of the control group.Conclusion pcDNA4 HisMaxA-hiL10,the hIL-10 efficient eukaryotic expression vectors,has been suecessfully constructed.
