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鸭白细胞介素-2基因在毕赤酵母中的表达
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张欣1，崔敏2，罗满林1*，贺东生1，黄毓茂1，房红莹1，陈钜豪1，马魁1
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(1.华南农业大学 兽医学院，广东 广州510642；2.福建农林大学 动物科学学院，福建 福州350002)
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摘要：
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根据GenBank中登录的鸭IL-2基因序列设计并合成了1对特异性引物，以经ConA诱导的广州鸭外周血淋巴细胞提取的总RNA为模板，用RT-PCR方法扩增出长度为360bp的目的基因片段,并将该基因克隆到pMD18-T载体上。酶切鉴定、PCR鉴定及序列测定结果表明，获得了鸭IL-2成熟蛋白基因的完整克隆。测序结果表明，该成熟蛋白基因由360个核苷酸组成，共编码119个氨基酸。克隆的鸭IL-2基因与GenBank上序列号为AY173028及AF294322的鸭IL-2基因的同源性高达99.4%。将目的基因克隆至真核表达载体pPICZαC上，构建了重组质粒pPICZαC-DuIL-2。酵母转化子经甲醇诱导发酵分泌表达了鸭IL-2基因。SDS-PAGE分析证实，鸭IL-2基因在毕赤酵母(Pichia pastoris)中表达成功，表达的重组蛋白的分子质量约为14.3ku。 |
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关键词：
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鸭；白细胞介素-2基因；毕赤酵母；表达 |
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中图分类号：Q 511:Q 786 文献标识码：
A 文章编号：1673-4696(2008)03-0229-04 |
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Expression of duck interleukin-2 gene in Pichia pastoris |
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ZHANG Xin1,CUI Min2,LUO Man-lin1 ,HE
Dong-sheng1,HUANG Yu-mao1,
FANG Hong-ying1,CHEN Ju-hao1,MA Kui1 |
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（1.College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642，China;
2.College of Animal Science,Fujian Agricultural and Forestry University,Fuzhou 350002，China） |
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Abstract: Based on the sequence of interleukin-2(IL-2) gene in GenBank,a pair of primers was designed to amplify the IL-2 mature protein gene from Guangzhou duck(DuIL-2) by RT-PCR with the total RNA prepared from peripheral blood lymphocyte induced by ConA as templates.The RT-PCR product was approximately 360bp in size.The DuIL-2 mature protein gene was cloned into the pMD18-T simple vector.Identification by enzymatic digestion,PCR amplification and sequencing indicated that the DuIL-2 mature protein gene was cloned successfully.The result demonstrated that the cloned gene was consisted of 360 nucleotides,encoding 119 amino acids.The cloned gene shared 99.4% identity with those ones available in GenBank (AY173028 and AF294322).The target gene was cloned into pPICZαC vector,and the recombinant plasmid pPICZαC-DuIL-2 was constructed.The expression of rDuIL-2 was induced with met-hanol.SDS-PAGE analysis showed that the rDuIL-2 protein was secreted successfully in the culture medium by Pichia pastoris strain X33 and it was 14.3ku in molecular mass. |
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Key words: duck;interleukin-2 gene;Pichia pastoris;expression |
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