[Screening of the clones efficiently expressing rhIL-12 in CHO cells and functional analysis of the expressed rhIL-12].
OBJECTIVE
To transfect the constructed recombinant human IL-12 (rhIL-12) eukaryotic expression plasmid (pcDNA6/v5-his-p70) into CHO cells and screen the efficiently and stably expressed clones; and to identify the biological activities of rhIL-12.


METHODS
pcDNA6/v5-his-p70 was transfected into CHO cells using polyethyleneimine (PEI) and positive clones were screened with Blasticidin as well as single clone amplification. Then the expression clone was identified by RT-PCR and the rhIL-12 expression of each clone was measured using ELISA. Finally, biological activities of the expressed rhIL-12 were analyzed with proliferation of lymphocytes in vitro and intracellular cytokine staining(ICS) . The stability of the selected clones was observed as well.


RESULTS
The RT-PCR results showed that a specific fragment of 1 800 bp could be amplified in all positive clones, among which six clones had rather high expression of 312.69 ng/L-719.10 ng/L with the highest expression of 719 ng/L (5x10(4) per cells, 48 h) by ELISA test. Biological activities analysis indicated that the expressed rhIL-12 could significantly increase the percentage of CD4(+)IFN-gamma(+) and CD8(+) IFN-gamma(+) cells in normal PBMC as well as the proliferation of PBMC to PHA/HCMV stimulation. The efficient expression of positive clones still remained stable after 6 months under maintenance of Blasticidin (2 ng/L).


CONCLUSIONS
The constructed human recombine hIL-12 eukaryotic expression vector can express stably with high efficiency in CHO cells and the expressed products show expected biological functions.
Qingdao University, China. lr0436@163.com
Rui
R
Lu
Wen-qing
WQ
Zhang
Hong
H
Yu
Dan
D
Li
D000818:Animals; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D011994:Recombinant Proteins; D018664:Interleukin-12