title: Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice.
abstract: Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-1,4-xylanase activities) from mollusca, Ampullaria crossean and a β-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.
authors: Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China, miaorongh@yahoo.com.cn.
authors: Miaorong
authors: M
authors: Huang
authors: Zicong
authors: Z
authors: Li
authors: Xiaoling
authors: X
authors: Huang
authors: Wenchao
authors: W
authors: Gao
authors: Cailin
authors: C
authors: Zhu
authors: Hui
authors: H
authors: Xu
authors: Yujuan
authors: Y
authors: Yuan
authors: Liang
authors: L
authors: Shuai
authors: Ruiai
authors: R
authors: Chen
authors: Zhenfang Wu
authors: Dewu Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D016466:CHO Cells / Q000201:enzymology; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D003412:Cricetulus; D051379:Mice; D008822:Mice, Transgenic; D008974:Mollusca / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase