Studies On The Expression Of Medical Proteins Hil-28a And Hgm-csf In Transgenic Silkworm, Bombyx Mori

Interleukin -28A, discovered by Kotenk and Sheppard in the year of 2003, was recently described as a member of a new cytokine family that shares the same function of antivirus, antitumor, and immunoregulation with type I interferon (IFN); The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is an important kind of the glycoprotein that has the immunity adjustment function, it plays the vital role in hemopoiesis regulation and the immunity adjustment, and has the important clinical value.To explore the possibility of foreign gene expression in transgenic silkworm mediated by non-transposon vector, a hIL-28A gene was inserted into the insect cells expression vector pIZT-V5-His to generate recombinant vector pIZT/V5-His-hIL-28A, the vector was transferred into silkworm eggs by sperm mediated gene transfer. Transgenic silkworms were obtained after being screened for gfp gene and verified it by PCR and Dot blot hybridization. A specific band with the molecular weight of 25 kD could be detected in transgenic silkworm by Western blotting using an goat anti-hIL-28A antibody, and the content of hIL-28A in the G3 generation transgenic silkworms estimated by ELISA was approximately 0.198, 0.32,0.238 ng per gram of freeze-dried in whole bodies, posterior silk glands and fat bodies, respectively. These results suggested that a heterologous gene could be integrated into silkworm genome by non-transposon vector and expressed successfully.Otherwise, to study the genetic stability of transgenic silkworms, the recombination protein activity and the effect by oral administration, transgenic silkworms obtained previously were reared from the fourth generation to the twelfth, each generational transgenic silkworms genomic DNA was respectively extracted and was identified by PCR amplification. The result verified the existence of the gfp, the neo, and hGM-CSF genes in the amplification from the G4 to G12 generation of silkworm. The ELISA measurement showed that the expression level of hGM-CSF was 20.8 ng per gram of freeze-dried silk gland in G11 transgenic silkworm silk gland, and was 5.15 ng per gram of freeze-dried silk gland in G3 transgenic silkworm silk gland. When cultured in 1640 culture medium mixed with posterior silk gland dissolved liquid, the k562 cells proliferated fast. Furthermore, leukopenia mice were oral administrationed with posterior silk gland dissolved liquid and theirs white blood cells increased. All these results suggested that transgenic silkworms have some genetic stability to express the recombination protein continuously, and it could be oral administrationed to increase white blood cells. The study provide basis for the expression of oral administrationed medical protein in transgenic Silkworm.