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[1]田汶佳,刘炯宇,江建平.无指盘臭蛙皮肤抗菌肽Odorgrin A真核表达载体的构建与转化[J].应用与环境生物学报,2010,16(02):222-227.[doi:10.3724/SP.J.1145.2010.00222]
 TIAN Wenjia,LIU Jiongyu,JIANG Jianping.Construction of Eukaryotic Expression Vector of Antimicrobial Peptide Odorgrin A from Odorrana grahami and Its Transformation into Pichia pastoris[J].Chinese Journal of Applied & Environmental Biology,2010,16(02):222-227.[doi:10.3724/SP.J.1145.2010.00222]
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无指盘臭蛙皮肤抗菌肽Odorgrin A真核表达载体的构建与转化()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
16卷
期数:
2010年02期
页码:
222-227
栏目:
研究论文
出版日期:
2010-04-25

文章信息/Info

Title:
Construction of Eukaryotic Expression Vector of Antimicrobial Peptide Odorgrin A from Odorrana grahami and Its Transformation into Pichia pastoris
文章编号:
200902030
作者:
田汶佳刘炯宇江建平
(1中国科学院成都生物研究所 成都 610041)
(2中国科学院研究生院 北京 100049)
Author(s):
TIAN WenjiaLIU JiongyuJIANG Jianping
(1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)
(2Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
关键词:
抗菌肽Odorgrin ApPIC9K毕赤酵母GS115转化无指盘臭蛙
Keywords:
antimicrobial peptide Odorgrin A pPIC9K Pichia pastoris GS115 transformation Odorrana grahami
分类号:
Q959.530.6 : Q78
DOI:
10.3724/SP.J.1145.2010.00222
文献标志码:
A
摘要:
根据无指盘臭蛙(Odorrana grahami)皮肤抗菌肽Odorgrin A的氨基酸序列,合成了以酵母偏爱密码子编码的Odorgrin A基因片段. 目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后,与经同样限制酶酶切的pPIC9K载体连接而成表达载体pPIC9K-Odo A. PCR扩增、酶切及测序检测的结果表明表达载体构建成功. 线性化的pPIC9K-Odo A经电击法转化毕赤酵母(Pichia pastoris)GS115宿主菌,营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测的结果表明,表达载体pPIC9K-Odo A成功地转化并整合入酵母基因组. 用甲醇对具遗传霉素G418高抗性的Odorgrin A重组酵母菌进行诱导表达,取酵母发酵液上清经SDS-PAGE电泳检测,结果初步表明,整合进酵母基因组的抗菌肽Odorgrin A基因已获得分泌表达,表达产物相对分子质量为3×103~4×103,与理论值相近. 图10 参17
Abstract:
A DNA fragment based on the amino acid sequence of Odorgrin A, antimicrobial peptide from skin of Odorrana grahami, was designed using yeast biased codons and synthesized chemically. The DNA fragment and vector pPIC9K were firstly digested by Xho Ι and EcoR Ι respectively, then ligated together. It was testified that the ligation product, expression vector named pPIC9K-Odo A, was successfully constructed based on PCR, enzymatic digestion, and DNA sequencing. The linearized pPIC9K-Odo A was transformed into Pichia pastoris GS115 strain by electroporation and homologously integrated with P. pastoris genome, which was proved by auxotrophic screening, geneticin resistant screening, PCR, and DNA sequencing. The positive colony with the highest geneticin resistance was induced by methanol. The SDS-PAGE analysis on the induced product detected a band of 3×103~4×103. It preliminarily indicated that the Odorgrin A had been successfully expressed in P. pastoris. Fig 10, Ref 17

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备注/Memo

备注/Memo:
四川省科技厅项目(Nos. 2008JY0001, 2006Z08-006)资助,国家自然科学基金项目(No. 30800100)和中国科学院知识创新工程青年人才领域前沿项目(No. CIB-2007-LYQY-01)部分资助 Supported by the Science and Technology Department of Sichuan, China (Nos. 2008JY0001, 2006Z08-006), and partially supported by the National Natural Science Foundation of China (No. 30670245) and the Knowledge Innovation Program of the Chinese Academy of Sciences (No. CIB-2007-LYQY-01)
更新日期/Last Update: 2010-04-20