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.
出处
《中国畜牧兽医》
CAS
北大核心
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)
