| EPO (erythropoietin) is a kind of glycoprotein that synthesized and secreted by kidney cells mainly in mammals and other higher eukaryotes. The basic physiological function of EPO is stimulating erythroid progenitor cells to differentiate into erythrocytes. Also, EPO can act as a protector of nerve cells and retina cells. EPO is clinical important because of its therapeutic role in the treatment of anemia caused by various disease and it has enormous officinal value. The produce of EPO mostly depended on protein expression system of mammal cell, such as CHO cell line. But the high cost limited the use of EPO. It's necessary to find a new expression system.Saprophytic fungus Trichoderma reesei has the ability of synthesizing and secreting protein, especially cellulases and hemicellulase. The production of cellobilhydrolase I (cbhl) in several high-yield strains can be produced up to 20-40 g per liter and cbhl promoter is considered as strong promoter. Trichoderma reesei has the advantages of simple structure, fast growth, and easy to manipulate and culture. And as a kind of eukaryotes, it also has the similar eukaryotic protein modification apparatus and post-translation modification mechanism to mammalian system. Trichoderma reesei has already been used as the host of heterologous protein. But the glycosylation extent affects the activity and stability of glycoprotein especially therapeutic proteins. In recent years, a start has been made to modifiy the glycosylation pathway in Trichoderma reesei to get strains that show a more mammalian-like type of glycosylation for the produce of human-raised glycoprotein.The factors that affect the production of protein include the promoter and its copy number, signal peptide and the process of protein isolation and purification. A high-level expression vector (pE) used to express the human-raised erythropoietin in Trichoderma reesei was constructed with the modified cbhl cis-acting element (containing CCAAT region,Ace2 binding sites and XlnR bindig sites) 4-copy promoter and the terminator of cbhl. The cbhl secretion signal peptide was added at the 5'-terminal of epo cDNA to help the expressed rhEPO secrete into extracellular culture. T. reesei protease deficient mutant Rut C30 U4 and T108 which had already been transformed into N-acetylglucosaminyltransferase I gene based on Rut C30 U4 were co-transformated with plasmids pE and pAN7-l. And about 78 and 150 transformants were isolated from the minimal medium containing 150 mg/L hygromycin B respectively. Two transformants with stable heredity of the epo gene were selected by PCR. The two transformants from Rut C30 U4 and T108 was named T47 and T112 respectively. The dot blot result showed that the epo gene was integrated into the chromosome of T47 and T112. The RT-PCR, SDS-PAGE and Western Blot results indicated the epo gene was transcripted and translated successfully and the fusion protein produced by T112 had a higher molecular weight than that of T47. The result indicated that the glycoprotein produced by N-acetylglucosaminyltransferase I gene transformed strain T112 had a further glycosylation extent. The erythropoietin produced by T47 was about 97.3 mg per liter and the erythropoietin produced by T112 was about 46.7 mg per liter.In this study, two epo expression vectors which containing N-terminal 6His-tag or C-terminal 6His-tag was constructed with cbhl cis-acting element 4-copy promoter and the 1.5kb-long terminator of cbhl as homologous domain and named plten and pltec respectively. T. reesei T108 was co-transformated with plasmids plten and pAN7-l or pltec and pAN7-1 and obtained 40 and 43 transformants respectively. Two transformants named N5 and C10 with stable heredity of the epo gene from plten and pltec were selected by PCR. Other analysises are still under going.This study provided that the human-raised glycoprotein could successfully expressed in filamentous fungus T. reesei. It's a new attempt to produce human-raised therapeutic proteins.
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