Expression,Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata
Abstract: Two cDNA fragments encoding human interleukin一12(hII一12)P35 and P40 subunits were isolated by RT.PCR from KB cells stimulated with PDBu,and then cloned into pCR .1 respcetively and down the double promoters of pAcUw 5 1.The recombinant baculovirus Ac—hiL2 was obtained by cotransfecting with pAeUW 5 1一ILl2 and baeuloGold M Linearized baculovirus DNA.Argyrogramma agnata larvae were infected with recombinant baculovirus Ac—hiLl2 by hemoeoel injection.The rhII, 1 2 was purified from the supernatant with affinity chromatography.The blood lymph supernatant har— vested and rhII,12 purified were SUbjected to SDS-PAGE(silver stain)and Western blot.The level of rhIL-12 was detected by EU SA.Bioactivity of purified rhII,12 samples were detected by M 1vr method.It is indicated that Ae-hlL12 can replicate in fat body and midgut cells of Argyrogramma Agnata larvae.The MW of rhlL一12 expressed was 75kD.The expresion levels of rhIL-12 were 17.8 /~g/lO cells and 200—300mg/L in Sf9 cells and Argyrogramma agnata Staudinger larvae blod lymph respectively.The purified rhII,12 has significant activities which enhanced NK eytotoxieity and increased the proliferation of human PBMC PHA-P activated significantly.