[High expression of human EPO gene in the larvae and pupae of the silkworm, Bombyx mori].
Erythropoietin (EPO) is a glycoprotein hormone produced primarily by the kidney, and is principal factor regulating red blood cell poduction. The synthesized EPO cDNA was inserted into the transfer vector pBlueBac III to generate the recombinant transfer plasmid pBlueBacEPO. Cotransfection of BmN cells with pBlueBacEPO DNA and Wild BmNPV DNA generated the recombinant virus rBmNPVEPO carrying EPO gene driven by the strong promoter of AcNPV polyhedrin gene. The results of Southern blot and PCR reaction confirmed that EPO gene had been correctly inserted in the target position in the BmNPV genome. ELISA assay showed that the EPO gene was expressed with high level in the larvae and pupae of the silkworm. The larvae and pupae produced as high as 62,800 u and 74,000 u in 1 mL hemolymph on the 4th day (larve) and the 5th day(pupae) after infection with the recombinant virus rBmNPVEPO, respectively. Western blot analysis showed the molecular weight of rhEPO produced in the lavae or pupae was about 26 kD. Biologic assay showed the rhEPO had high activity (about 63,000 u in per milliliter of hemolymph) in vitro.
Zhang
C X
CX
Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Jiang
Y L
YL
Hu
C
C
Wu
X F
XF
D000818:Animals; D012831:Bombyx / Q000235:genetics*; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007814:Larva / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D004921:Erythropoietin