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| Cloning of a β-glucosidase Gene (bgl1) from Asperjillus niger and Expression in Chinese Hamster Ovary (CHO) Cells |
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Abstract β-glucosidase is one of the three members of the cellulase system and is considered to be the rate-limiting enzyme of cellulose saccharification. The objective of the study was to clone the β-glucosidase gene (bgl1) from Asperjillus niger, and analyze its expression in Chinese hamster ovary (CHO) cells. The DNA fragment was amplified by PCR from Asperjillus niger, and then cloned into pMD18-T vector. A DNA fragment containing the coding sequence of pig parotid secretory protein signal peptide (sp) and bgl1 was assembled by SOE-PCR and subcloned into the expression vector pcDNA6/His A using BamHⅠand XhoⅠrestriction sites to generate pc6-sp-bgl1. To evaluate transgene expression, plasmid DNA was purified and transiently transfected into CHO cells, then RT-PCR and Western blot analyses were used and the enzymatic activity was determined by DNS(dinitrosalicylic acid). Sequence analysis showed that the DNA sequence and the putative amino acid sequence shared 91% and 99% identity with the reported sequences, respectively. RT-PCR and Western blot analyses showed that the exogenous gene bgl1 was expressed and recombinant protein was secreted into cell culture medium. The enzymatic activity of culture supernatants from 48 h-transfected cells to D-(-)-salicin was 1.74 U/mL, indicating that the recombinant protein with the activity of β-glucosidase. We cloned the β-glucosidase gene bgl1 from Asperjillus niger and expressed the gene in CHO cells. The study paves the way for further research of β-glucosidase in monogastric animals.
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Received: 21 May 2012
Published: 26 November 2018
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