摘要
用PCR方法以Ⅵ型胶原蛋白基因组DNA为模板,获得Ⅵ型胶原蛋白α2链基因,并将该基因插入到表达载体pPIC9K,对重组质粒pPIC9K-COL6A2所含的目的片段进行双向测序。将测序正确的重组质粒用限制性内切酶SalⅠ线性化,经电击转化到毕赤酵母GS115感受态细胞中。通过MM/MD法进行甲醇利用表型的筛选,PCR鉴定目的基因的整合及G418梯度筛选高拷贝转化菌。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,目的蛋白分泌表达到胞外。1%甲醇诱导72h的发酵液,经SDS-PAGE电泳鉴定该重组蛋白分子质量约为32kD。
The collagen Ⅵ chain α2 gene was amplified from human ollagen Ⅵ genomic DNA and subcloned into the vector of pPIC9K,and verified by DNA sequencing.The resultant recombinant plasmid pPIC9K-COL6A2 was digested by SalⅠ and transformed into the competent celss of Pichia pastoris strain GS115 through electroporation.Mut（methanol utilization）＋ and Muts transformants were screened with MM and MD plates.Clony PCR was used to analyze Pichia pastoris integrants in order to determine if the gene of interest has been integrated into the Pichia pastoris genome.Cells with stable expression of collagen were screened in a medium containing G418.Regulated by α-factor,AOX1 gene promoter and termination signal of yeasts,the recombinant collagen was expressed and secreted from the cells.After induction with 1% methanol,SDS-PAGE analysis showed that the molecular weight of recombinant collagen was approximately 32 kD.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2013年第9期224-227,共4页
Food Science
基金
吉林省科技厅自然科学基金项目(201115189)
