食品科学 ›› 2013, Vol. 34 ›› Issue (9): 224-227.doi: 10.7506/spkx1002-6630-201309046

• 生物工程 • 上一篇    下一篇

Ⅵ型类人胶原蛋白基因COL6A2的克隆及在毕赤酵母中的分泌表达

徐立群,王 皓,孙 旸,王 刚,陈 欢,陈 光*   

  1. 吉林农业大学 生物物理实验室,吉林 长春 130118
  • 收稿日期:2013-01-05 修回日期:2013-04-20 出版日期:2013-05-15 发布日期:2013-05-07
  • 通讯作者: 陈 光 E-mail:245137993@QQ.com
  • 基金资助:

    吉林省科技厅自然科学基金项目 (201115189)

Cloning of Collagen Ⅵ COL6A2 and Its Expression in Pichia pastoris

XU Li-qun ,WANG Hao,SUN Yang,WANG Gang,CHEN Huan,CHEN Guang*   

  1. Laboratory of Biophysics, Jilin Agricultural University, Changchun 130118, China
  • Received:2013-01-05 Revised:2013-04-20 Online:2013-05-15 Published:2013-05-07
  • Contact: CHEN Guang E-mail:245137993@QQ.com

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。

关键词: 胶原蛋白, pPIC9K, 毕赤酵母

Abstract:

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.

Key words: collagen, pPIC9K, Pichia pastoris

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