Expressing Of HIGF-Ⅰ In Silk Gland Bioreactor Of Transgenic Silkworm

The silk gland of silkworm, as a bioreactor with the basic characteristics, was an important organ for silk protein synthesizing and secreting. To express human insulin-like growth factor-I (hIGF-I) in transformated Bombyx mori cultured cells and silk gland, the transgenic vector pigA3GFP- Ser-hIGF-ie-neo was constructed with a neomycin resistance gene driven by baculovirus ie-1 promoter, hIGF-I gene which under control of silkworm sericin promoter Ser-1. We succeeded to select stable transformation of BmN cells expressing hIGF-I by using antibiotic G418 after BmN cells were transfected with the piggyBac vector and the helper plasmid. The expression level of hIGF-I determined by ELISA was about 7.8×103 pg pg in 5×105 cells. The transgenic vector pigA3GFP-Ser-hIGF-ie-neo was transferred into the eggs by using sperm-mediated gene transfer. Finally, we obtained 2 transgenic silkworms under the screening of neo and gfp genes, and identified with PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2.44×103 pg in each gram of middle silk gland of transgenic silkworm of G1 generation. Besides, another transgenic vector pigA3GFP- Fhx/P25-hIGF-ie-neo was constructed with a neomycin resistance gene driven by ie-1 promoter, hIGF-I gene which under control of silkworm fibroin promoter Fhx/P25. The transgenic vector was also transferred into the eggs by using sperm-mediated gene transfer. We obtained transgenic silkworm aftere being screening and deteced by molecular biology method .Exprssiong lever of hIGF-I was 152×103pg in each gram of posterior silk gland of transgenic silkworm of G1 generation by ELISA.