[Construction and expression of the eukaryotic plasmids containing different subunits of human IL-12 gene].
OBJECTIVE
To clone and construct the eukaryotic expression plasmids containing different subunits of human IL-12 and to investigate its expression following transient transfection into COS-7 cells.


METHODS
Human leukaemia cells (HL-60 and THP-1) were stimulated by DMSO, IFN-gamma and LPS, and the target genes including IL-12 p40 and p35 subunits were amplified by RT-PCR. P40 and p35 fragments were then connected and the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 were constructed by double restriction enzymes digestion and connection. Positive plasmids were transiently transfected into COS-7 cells with siPORT XP-1 reagent. The expression of target gene was detected by RT-PCR and ELISA.


RESULTS
The recombinant plasmids were confirmed by PCR amplification, double restriction enzymes digestion and DNA sequencing. Target gene expression was detected by RT-PCR and ELISA in the transfected COS-7 cells.


CONCLUSIONS
The successful construction of the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 provides a useful tool for further research on the immune regulatory role of IL-12.
Hao
Mu
M
Department of Infection and Immunity, Preclinical and Forensic Medicine School of West China Medical Center, Sichuan University, Chengdu 610041, China. haomusg@163.com
Bao
Lang
L
Zhang
Hui-Dong
HD
Gao
Lei
L
Li
Ya-Sha
YS
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D021122:Protein Subunits; D018664:Interleukin-12; D004262:DNA Restriction Enzymes