黑曲霉木聚糖酶基因xynA的克隆及真核表达载体的构建

Cloning of Xylanase Gene xynA from Aspergillus niger and Construction of Eukaryotic Expression Vector

从黑曲霉(Aspergillus niger)GI M3.452中克隆得到木聚糖酶基因xynA的成熟肽编码序列。通过重叠延伸PCR将不带原基因信号肽编码序列的xynA基因片段和猪腮腺分泌蛋白(parotid secretory protein,PSP)基因的信号肽进行拼接得到融合片段SPA,并将其克隆到真核表达载体pcDNA6/HisTMA中,得到重组质粒pcDNA-SPA,重组质粒经过酶切、测序鉴定其读码框的正确性,在脂质体介导下将重组质粒pcDNA-SPA转染猪肾细胞(PK15),通过RT-PCR证实其在PK15细胞中表达,并在细胞培养液中检测到木聚糖酶活性为7.6I U/mL。

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/HisTM A.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.