RESUMEN
To develop the stable transformants of the silkworm (Bombyx mori) BmN cells that could continuously express the exogenous gene based on a non-transposon vector, an expression cassette containing human granucyto-macrophage colony-stimulating factor (hGM-CSF) gene driven by ie-1 promoter from B. mori nucleopolyhedrovirus was inserted into pIZT-V5-His to form a recombinant vector pIZT-IE-hGM-CSF, followed by transfecting the constructant into BmN cells, the stable ie-hGM-CSF cell lines were obtained after being selected with Zeocin. PCR result using the genomic DNA of the transformed BmN cells as template illustrated a specific fragment of ie-hGM-CSF, and Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the transformed cells, meanwhile, the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 10(6) transformed BmN cells.
Asunto(s)
Animales , Humanos , Animales Modificados Genéticamente , Bombyx , Biología Celular , Genética , Metabolismo , Línea Celular , Fibroínas , Genética , Vectores Genéticos , Genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Genética , Proteínas Recombinantes de Fusión , Genética , Transformación GenéticaRESUMEN
Hydrogen production from lignocellulosic biomass is both sustainable and environmentally friendly, which is garnering more and more attention across the world, with an expectation to challenge the shortage of fossil fuels supply and climate change as well. In this article, the update research progress and technology development of biohydrogen production are reviewed, with a focus on biomass pretreatment, hydrogen-producing microorganisms and process engineering strategies. And in the meantime, a roadmap for more efficient and economic biohydrogen production is envisioned.
Asunto(s)
Bacterias , Metabolismo , Fuentes de Energía Bioeléctrica , Microbiología , Biomasa , Biotransformación , Fermentación , Hidrógeno , Metabolismo , Lignina , MetabolismoRESUMEN
Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.