The mature peptide coding sequence of xylanase gene xynA was amplified by RT-PCR from Aspergillus niger GIM3.452 total RNA extracts.The result suggested that the mature peptide sequence of xynA was consisted of 927 bp,and encoded 308 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).SPA was subcloned into the eukaryotic expressing plasmid vector pcDNA6/HisTMA.The recombinant plasmid pcDNA-SPA was identified by PCR,enzyme digestion and DNA sequencing.The result showed that the recombinant plasmid of pcDNA-SPA was constructed correctly.Meanwhile,the PK15 cells were transfected with pcDNA-SPA 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 7.6 IU/mL.
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张 茂,刘德武,林 纯,贺晓燕,孟繁明,周庆丰,杜云平,吴珍芳. Cloning of Xylanase Gene xynA from Aspergillus niger and Construction of Eukaryotic Expression Vector[J]. Jorunal of Huazhong Agricultural University,2010,29(5):588-592.