黑曲霉β-甘露聚糖酶基因manA在PK15细胞中的分泌表达

Secretion Expression of β-mannanase(manA)Gene in PK15 Cells from Aspergillus niger

通过RT-PCR方法,从黑曲霉总RNA中克隆出β-甘露聚糖酶基因manA的成熟肽编码序列。将其与猪腮腺分泌蛋白(parotid secretory protein,PSP)基因的信号肽序列通过重叠延伸PCR方法得到拼接片段,并插入到真核表达载体pcD-NA6.0/HisTMA中,得到重组质粒pcDNA-PSmanA。重组质粒经过PCR、酶切、测序鉴定,证实含有目的片段,且读码框完全正确。在脂质体介导下将pcDNA-PSmanA转染猪肾(PK15)细胞进行分泌表达,通过RT-PCR方法证实其在PK15细胞中表达,并在细胞培养液中检测酶活性得到β-甘露聚糖酶基因酶活性为14.5 IU/mL。

The mature β-mannanase gene（manA） was amplified by RT-PCR from Aspergillus niger total RNA.Then,the mature peptide sequence and signal peptide sequence of pig parotid secretory protein（PSP）gene were splicing by overlap extension PCR（SOE-PCR）,which was subcloned into the eukaryotic expressing plasmid vector pcDNA6.0/HisTMA.The recombinant plasmid pcDNA-PSmanA was identified by PCR,enzyme digestion and DNA sequencing.The result showed that the recombinant plasmid of pcDNA-PSmanA was constructed correctly.Meanwhile,the PK15 cells were transfected with pcDNA-PSmanA by cationic liposome,and the mRNA of the target gene was determined by RT-PCR.The maximum yield of the recombinant β-mannanase in cell culture medium was 14.5 IU/mL.