[Expression of murine interleukin 12(mIL-12) in insect cells].
Since human IL-12 is species-specific in its functions and elicits little biological responses from mouse lymphocytes, it is necessary to express recombinant murine IL-12 for the usage in studying the effects of this cytokine in various rodent models. Thereby, we can investigate the role of IL-12 in immune response in vivo and evaluate its potential clinical utility. Thus, we firstly constructed two expression vectors, pVL1393-mp40 and pVL1393-mp35. They were used to co-transfect the insect cells(Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-mp40 and AcNPV-mp35 were visually screened out, and mp40 and mp35 were co-expressed in the insect cells co-infected by AcNPV-mp40 and AcNPV-mp35. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the recombinant mIL-12 was expressed successfully in the insect cells. The molecular weights of recombinant mp40 and mp35 were 40 KDa and 22 KDa on SDS-PAGE under reducing conditions, respectively. The apparent molecular weight of recombinant mIL-12 is 80 KDa under non-reducing conditions of Western blot. Biological activity of the recombinant product was detected in conditional medium using antibody-capture bioassay. The expression level of recombinant mIL-12 was about 10-15 micrograms/10(6) cells, as compared with the calibration curve of mIL-12.
Li
C L
CL
Shanghai Institute of Cell Biology, Academia Sinica, Shanghai 200031.
Shen
H
H
Lin
M Q
MQ
Zhang
Z L
ZL
Shen
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QX
D000818:Animals; D018664:Interleukin-12 / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*
D011994:Recombinant Proteins; D018664:Interleukin-12