[Construction, eukaryotic expression and biological activities of a recombinant human single chain interleukin-12 fusion gene].
OBJECTIVE
To explore the feasibility of recombinant human interleukin 12 (IL-12) with biological activities by molecular biological techniques.


METHODS
Both p40 and p35 subunits cDNA of human IL-12 were cloned from mRNA extracted from NC-37 cell line by using RT-PCR, and the fusion gene (p40-linker-p35) of recombinant human single chain IL-12 (rhscIL-12) was constructed by using a polypeptide linker (Gly(4)Ser)(3). rhscIL-12 eukaryotic expressing vector pcDNA3.1 (+)-hscIL-12 was constructed by inserting the rhscIL-12 fusion gene into pcDNA3.1 (+) eukaryotic expressing plasmid. COS-7 cells were transfected with pcDNA3.1 (+)-hscIL-12 plasmid.


RESULTS
A stable rhscIL-12 expressing cell line COS-rhscIL-12 was obtained by G418 selection. Western blot showed the presence of a 70 x 10(3) band of the fusion protein, which specifically bond to mouse-anti-human IL-12 monoclonal antibody. The assays of biological functions showed that the fusion protein had strong bioactivities in stimulating the lymphocyte proliferation, enhancing the NK cell cytotoxicity and increasing the IFN-gamma production.


CONCLUSIONS
The constructed rhscIL-12 fusion gene could express biologically functional rhscIL-2 fusion protein.
Zhang
M
M
Institute of Hematology, the First Affiliated Hospital of Xian Medical University, Xian 710061, China.
Si
L
L
Jin
L
L
Wang
Y
Y
Geng
Y
Y
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism; D020014:K562 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12