Cloning Of Endo-β-1,4-d-glucanase Gene From Aspergillus Fumigatus And Its Expression In Pichia Pastoris

【Objective】 As the research object to the fumigatus B-2-3, With morphology and ITS sequences identified of the strain; By clonied endo cellulase encoding genes and inserted into Pichia pastoris expression vector secreted p PIC9 K and through the high-voltage turn introduced into Pichia pastoris secreted expression, and recombinant strain was measured activity,in order to provide the appropriate theoretical basis to struction of recombinant bacteria and cellulase transformation in the years to come; 【Method】Observed on the production of cellulase strain B-2-3 morphological,and in terms of the known fungal proven design primers, as the produce cellulase strain B-2-3 of DNA a template to amplify its ITS sequence and identify ITS sequences;the cellulose enzyme endonuclease gene sequence of Aspergillus fumigatus on Gene Bank known published reference sequence Aspergillus fumigatus(JQ796068.1), designed without signal peptide-specific primers, cloned Aspergillus fumigatus B-2-3 EG cellulase gene; recombinant p PIC9K-EG, with Bgl II restriction endonuclease enzyme digested and In turn electricity meter into pichia GS115, SDS-PAGE protein detection and purification of proteins by nickel column, then CMC-Na-Congo red plates and endo cellulase measured GS-9K-EG activity. 【Results】 The morphology and ITS sequence identification producing cellulase strains B-2-3 was Aspergillus fumigatus; endoglucanase gene was cloned into the inside of Aspergillus fumigatus B-2-3, the top length c DNA 1361 bp, encoding 437 amino acids predicted protein molecular size 46.40 KDa, belong to GH7. The cloned gene and Aspergillus fumigatus(JQ796068.1) without signal peptide gene sequence alignment results showed that in the first 158 bases have a different mutation at nucleotide C to A, but was same sense mutation, not impact on the expression of the protein; The eukaryotic expression vector p PIC9K-EG was constructed and expressed in Pichia pastoris GS115 successfully, the expression of the protein after purification was 3.86μg / μL, detected by the CMC-Na-Congo red tablet GS-9K-EG can produce hydrolysis circle, endo cellulase measured GS-9K-EG endo activity in cultured 72 h highest 8.09U/m L. 【Conclusion】(1) The the production of cellulase strains B-2-3 identified as a Aspergillus fumigatus;(2) EG gene was successfully cloned from Aspergillus fumigatus, and Expressed in yeast expression system successfully, with original strain B-2-3 as compared to the recombinant strain GS-9K-EG enzyme activity decreased significantly, which further illustrates the synergistic action principle cellulase gene, for the further study of the interaction of cellulase and its co-expression laid the theoretical foundation.