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| Cloning and eukaryotic secrete expressing Xylanase gene xynB from Aspergillus niger |
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Abstract Abstract：The mature peptide coding sequence of xylanase gene xynB was amplified by RT-PCR from Aspergillus niger GIM3.452 total RNA extracts. The result suggested that the mature peptide sequence of xynB is consisted of 567 bp,and encodes 188 amino acids. Then,the mature peptide sequence and signal peptide sequence of pig parotid secretory protein gene were splicing by overlap extension PCR (SOE-PCR). PSxynB was subcloned into the eukaryotic expressing plasmid vector pcDNA6/His A and transformed into host E. coli strain DH5a for identification. The recombinant plasmid pcDNA-PSxynB was identified by PCR, enzyme digestion and DNA sequencing. The result showed that the recombinant plasmid of pcDNA-PSxynB was constructed correctly.
Meanwhile,the PK15 cells were transfected with pcDNA-PSxynB by cationic liposome，and the mRNA of the target gene was determined by RT-PCR. The maximum yield of the recombinant xylanase in cell culture medium was 35IU/ml.
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Received: 05 August 2009
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