Expression of human interleukin 12 (hIL-12) in insect cells.
Human interleukin-12(hIL-12) is a herterodimer cytokine, which consists of two disulfide-linked subunits, p40 and p35. This paper reports the expression of hIL-12 using the Baculovirus Expression System in insect cells. First, we constructed two expression vectors pVL1392-hp40 and pVL1393-hp35, and then they were used to co-transfect the insect cells (Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-hp40 and AcNPV-hp35 were visually screened out. Biological activity of the recombinant hIL-12 (rhIL-12) was detected in the conditioned medium using proliferation assay of PHA-activated human lymphocytes and the expression level was about 1.5 approximately 2 microg/10(6) cells. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the subunits of rhIL-12, hp35, and hp40 were expressed successfully in the insect cells. The apparent molecular weights of rhIL-12 and hp40 homodimer were 76 kDa and 92 kDa under non-reducing conditions of Western blot, respectively. The recombinant hp40 can significantly inhibit the biological activity of hIL-12.
Shen
H
H
Shanghai Institute of Cell Biology, the Chinese Academy of Sciences.
Li
C
C
Lin
M
M
Zhang
Z
Z
Shen
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Q
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000502:physiology; D017924:Nucleopolyhedroviruses; D012333:RNA, Messenger; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018411:Spodoptera / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12