摘要
进行了组织型纤溶酶原激活剂(t-PA)突变体Reteplase(r-PA)基因的克隆,并在甲醇毕赤酵母(Pichia methanolica)中实现了胞外表达。以基因工程菌株里氏木霉(Trichoderma reesei)306染色体DNA为模板,通过PCR技术克隆了r-PA基因,将扩增产物克隆到pMD18-T载体上并进行了序列测定。测序结果表明:克隆到的基因序列长1.1 kb,与已发表的Reteplase基因同源性达99.91%,其编码的氨基酸顺序一致。文中还构建了甲醇毕赤酵母分泌性表达载体pMETαA-r-PA,用PacⅠ单酶切将pMETαA-r-PA线性化后,采用电转化的方法将其导入甲醇毕赤酵母PMAD16中,PCR和表型鉴定表明,筛选到Reteplase基因已经整合到甲醇毕赤酵母染色体上的阳性克隆。经摇瓶初步培养及以甲醇为唯一碳源诱导表达,胞外Reteplase表达水平最高达27.93mol/(s.L)。
The cloning of Reteplase （r-PA） gene of tissue-type plasminogen activator （t-PA） mutant was first carfled out, and the gene was extracellularly expressed in Pichia methanolica. Next, the gene-encoding Reteplase （r-PA） was cloned to pMD18-T vector by PCR using recombinant strain Trichoderma reesei 306 chromosome DNA as the template. Then, a sequence determination was conducted, indicating that the cloned gene sequence is of a length of 1.1 kb and an identity of 99.91% or 100% respectively with the published Reteplase gene or amino acids. Moreover, the secreted expression plasmid pMETαA-r-PA of Pichia methanolica was not only constructed and digested with Pac Ⅰ but also transformed into Pichia methanolica PMAD16 by means of electroporation. The positive transformants that integrate r-PA genes in their genomes were thus obtained, which was verified by PCR technique and Mut phenotype determination. After the shake-flask culture and the induced expression with methanol as the only carbon source, the extracellular Reteplase （r-PA） activity was up to 27.93 mol/（ s·L）.
出处
《华南理工大学学报（自然科学版）》
EI
CAS
CSCD
北大核心
2006年第12期25-29,50,共6页
Journal of South China University of Technology(Natural Science Edition)
基金
黑龙江省自然科学基金资助项目(D0352)
