宇佐美曲霉木聚糖酶在哺乳动物细胞中的分泌表达  

Aspergillus usamii Xylanase Secrete Expression in Mammalian Cells

作　　者：张茂[1] 邹娴[2] 林纯[2] 刘德武[2] 吴珍芳[2] 蔡更元[1] 

机构地区：[1]广东省农业科学院畜牧研究所畜禽育种国家重点实验室广东省动物育种与营养公共实验室,广东广州510640 [2]华南农业大学动物科学学院,广东广州510642

出　　处：《中国畜牧兽医》2012年第1期23-27,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家重大专项(2008ZX08006004;2009ZX08006-012B)

摘　　要：本试验旨在从宇佐美曲霉菌株GIM3.36中克隆得到木聚糖酶基因xyn的成熟肽编码序列(555bp),并将其克隆到真核表达载体pcDNA6/HisTM A中的不同位置,分别得到重组质粒pcDNA-spna和pcDNA-spnb,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定木聚糖酶酶活,结果显示,重组质粒pcDNA-spna转染细胞后表达的酶活力最高为8.53U/mL,较pcDNA-spnb表达的酶活(6.87U/mL)高24%,实现了微生物基因在哺乳动物细胞的分泌表达,为xyn基因在转基因方面的利用提供了依据。The mature peptide coding sequence of xylanase gene xyn（555 bp） was amplified by RT-PCR from Aspergillus niger GIM 3.36 total RNA extracts.It was subcloned into different locations of the eukaryotic expressing plasmid vector pcDNA6/HisTM A.The recombinant plasmid pcDNA-spna and pcDNA-spnb was identified by PCR,enzyme digestion and DNA sequencing.The result showed that the recombinant plasmids were constructed correctly.Meanwhile,the PK15 cells were transfected with pcDNA-spna and pcDNA-spnb by cationic liposome,and the mRNA of the target gene was determined by RT-PCR.The maximum yield of the recombinant xylanase（transfected pcDNA-spna） in cell culture medium was 8.53 U/mL and higher 24% the recombinant xylanase 6.87 U/mL（transfected pcDNA-spnb）.To achieve the purpose that microbiology secrete expression in mammalian cells and provided basis for using xyn gene to transgenic.

关 键 词：宇佐美曲霉 木聚糖酶 哺乳动物细胞 分泌表达 

分 类 号：Q78[生物学—分子生物学]