黑曲霉木聚糖酶基因(xynB)的克隆及真核分泌表达  

Cloning and Eukaryotic Secrete Expressing Xylanase Gene(xynB) from Aspergillus niger

作　　者：张茂[1,2] 周庆丰[1,2] 杜云平[1,2] 刘德武[1,2] 孟繁明[1,2] 周美华[1,2] 吴珍芳[1,2] 

机构地区：[1]华南农业大学动物科学学院,广州510642 [2]广东省温氏集团研究院,新兴527400

出　　处：《农业生物技术学报》2010年第2期368-373,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(2008AA101008);国家重大专项(2008ZX08006004)资助

摘　　要：通过RT-PCR方法,以黑曲霉(Aspergillus niger)GIM3.452总RNA为模板,克隆出木聚糖酶B(xy-lanaseB,xynB)基因的成熟肽编码序列(567bp),编码188个氨基酸。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(SOE-PCR)得到拼接片段PSxynB,并将其克隆到真核表达载体pcDNA6/HisTMA中,得到重组质粒pcDNA-PSxynB,重组质粒经过酶切、测序鉴定,证实含有目的片段,且构建正确。在脂质体介导下将重组质粒pcDNA-PSxynB转染猪肾细胞(PK15),通过RT-PCR证实其在PK15细胞中表达,并在细胞培养液中测到了木聚糖酶活最高达36.4IU/mL。The mature peptide coding sequence of xylanase gene（xynB） was amplified by RT-PCR from Aspergillus niger GIM3.452 total RNA extracts.The result showed that the mature peptide sequence of xynB was consisted of 567 bp, and encoded 188 amino acids.Then, the mature peptide sequence of xynB gene and signal peptide sequence of pig parotid secretory protein gene（PSP） were spliced by overlap extension PCR（SOE-PCR）, which PSxynB was obtained.PSxynB was subcloned into the eukaryotic expressing plasmid vector pcDNA6/HisTM A and transformed into host Escherichia coli strain DH5α, obtaining recombinant plasmid pcDNA-PSxynB.The pcDNA-PSxynB was identified by PCR, enzyme digestion and DNA sequencing.The result showed that the pcDNA-PSxynB was constructed correctly.Meanwhile, the porcine kidney cells（PK15） 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 36.4 IU/mL.

关 键 词：黑曲霉 木聚糖酶 分泌表达 

分 类 号：Q786[生物学—分子生物学]