Expression Of HGM-CSF In Transgenic Silkworms/Cells And Production Of Green Fluorescent Silk

The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a kind of hematopoietic stimulating factors, which can stimulate the proliferation, maturation and differentiation of T-cells and macrophages, and enhances the immune system. In the regulation of hematopoiesis and immune regulation plays an important role, and therefore has important clinical value and also occupies an important position in the international biotechnology pharmaceutical market.The silkworm (Bombyx mori) belonging to Lepidoptera Saturniidae, can be raised on a large-scale, genetic background is more clearly, and their silk glands have the powerful ability to synthesis and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk gland of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF -LPA-FibL-R was constructed, into was inserted a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven GFP. The targeting vector was then introduced into the eggs of silkworm by sperm-mediated gene transfer; and we got fluorescent silkworms from the G0 generation silkworms, and then the fluorescent silkworms that were verified by PCR and DNA hybridization after being screened for the gfp gene were the transgenic silkworm. Western blotting analysis indicated hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the silk glands of the G3 generation transgenic silkworms. ELISA assay indicated that the expression level of hGM-CSF was about 2 700 pg per gram of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced at the desired locus of the silkworm genome by gene targeting and expressed successfully. Meanwhile, replacing the region from the thirtieth nucleotide of exon 6 to the thirty-third nucleotide of exon 7 of the fib-L gene with exogenous DNA did not seem to affect production of silk and successful cocooning of the transgenic silkworms.In order to reform the silk by heredity molecule, this study, a targeting vector pSK-FibL-L-GFP-FibL-R was constructed, into was inserted the termination codon TAA of the fib-L upstream 1.2 kb and downstream 0.5 kb as homogenous arms, respectively. The GFP gene was cloned into the two homologous arms, so that the ORF of GFP consistent with the ORF of fib-L . The transgenic vector was then introduced into the eggs of silkworm by sperm-mediated, and then combined identification of molecular biology; we obtained the transgenic silkworm that could spit green fluorescent silk gene targeting. The relevant results for molecular design to change the properties of silk laid a foundation.In addition, we generated stable transformants of the silkworm (Bombyx mori) BmN cells continuously expressing a target gene from a non-transposon vector, a expression cassette containing human granucyto-macrophage colony-stimulating factor (hGM-CSF) gene driven by the ie-1 promoter of B.mori nucleopolyhedrovirus was inserted into pIZT-V5-His to generate recombinant vector pIZT-IE-hGM-CSF. The stably ie-hGM-CSF cells lines were selected by using the antibiotic zeocin after the BmN cells were tranfected with the vector. The specific fragment of ie-hGM-CSF could be amplified from the genomic DNA of the transformed BmN cells, the specific band of 22 kDa presenting hGM-CSF was detected in the transformed cells by western blot with the specific antibody, and the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 106 transformed BmN cells.