水蛭素基因毕赤酵母表达载体的构建及高拷贝稳定整合菌株的筛选
毕赤酵母,,水蛭素；毕赤酵母；基因重组,0引言,1材料和方法,2结果,3讨论,【参考文献】
(第四军医大学：1西京医院中医科，2西京医院检验科，3基础部药理学教研室，陕西 西安 710033)Construction of hirudin expression vectors and screening of stable multicopy integrants in Pichia pastoris
LONG Yin, LIU JiaYun, LIU Li, WANG ZongRen
1Department of Traditional Chinese Medicine, 2Department of Clinical Laboratory, Xijing Hospital, 3Department of Pharmacology, School of Basic Medicine, Fourth Military Medical University, Xi’an 710033 China
【Abstract】 AIM: To construct secretory hirudin variant I (HV1) expression vector and to obtain stable multicopy integrants in Pichia pastoris. METHODS: Based on the established amino acid sequence of HV1 and preferred codons of P. pastoris, optimal HV1 gene sequence was designed and chemically synthesized in 8 oligonucleotides. The whole HV1 gene was constructed with these nucleotides by phosphorylation, annealing, ligation and PCR amplification. Then the synthetic gene was sequenced and proved to be the same as the designed. The HV1 gene was fused to pPIC9 α factor signal and subcloned into pPIC9K secretory expression vector. The construct was linearized with SalI and transformed into P. pastoris strain GSM1168, and stable multicopy integrants were screened in medium containing different concentrations of G418. RESULTS: The secretory HV1 expression vector was successfully constructed and stable highcopy integrated strains were obtained. CONCLUSION: This result offers efficient P. pastoris strains for mass production of hirudin protein. ......
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