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| 鸭MHC-Ⅰα链胞外区成熟肽基因的克隆、序列分析及表达载体构建 |
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孙 凯1,2, 李新生1,2, 崔保安1,2, 陈红英1,2, 魏战勇1,2, 张 蕾1,2, 宋亚鹏1,2, 阮武营1,2
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1.河南农业大学 牧医工程学院;2.河南省动物性食品安全重点实验室
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| 摘要: |
| 【目的】克隆鸭MHC-Ⅰα链胞外区成熟肽基因cDNA序列，构建并鉴定其原核表达质粒pET/Du MHC-Ⅰ。【方法】依据GenBank/DDBJ/EMBL基因库中公布的鸭MHC-Ⅰ基因序列设计1对引物，采用RT-PCR技术从鸳鸯鸭脾脏组织中扩增MHC-Ⅰα链胞外区成熟肽基因cDNA，T-A克隆到pGEM-T Easy载体中，并进行测序。通过PCR和酶切的方法，将鸭MHC-Ⅰα链胞外区成熟肽基因亚克隆入pET-28a(+)载体，构建重组表达质粒pET/Du MHC-Ⅰ，并进行PCR、NcoⅠ和NotⅠ双酶切及测序鉴定。【结果】测序结果表明，获得了MHC-Ⅰα链胞外区成熟肽基因，其长度为813 bp，编码271个氨基酸。MHC-Ⅰα链胞外区成熟肽基因与GenBank中登录的鸭MHC-Ⅰα链胞外区成熟肽基因序列的同源性为89.4%～91.7%，氨基酸同源性为78.2%～83.7%；与鸡、鹅、鹌鹑、草鱼、狗、猪、鼠和人的MHC-Ⅰα链胞外区成熟肽氨基酸序列的同源性分别为64.6%，79.3%，59.4%，41.0%，26.6%，45.0%，12.5%和22.1%，这表明MHC-Ⅰα链胞外区成熟肽基因存在着种属多样性，且亲缘关系越近，同源性越高。所获重组表达质粒pET/Du MHC-Ⅰ经PCR、酶切、测序鉴定，证实鸭MHC-Ⅰα链胞外区成熟肽基因cDNA已正确克隆入pET-28a(+)表达载体。【结论】 成功克隆出鸭MHC-Ⅰα链胞外区成熟肽基因，并构建了重组表达质粒pET/Du MHC-Ⅰ。 |
| 关键词: 鸭 MHC-Ⅰα链 胞外区成熟肽基因 原核表达载体 |
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| 基金项目:国家“十一五”科技支撑计划专项(2006BAD06A08) |
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| Gene cloning,sequence analysis and prokaryotic expression vector construction of duck MHC-Ⅰα chain mature peptide of the extracellular domain |
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| Abstract: |
| 【Objective】 The study was done to clone duck MHC-Ⅰα chain mature peptide gene cDNA sequence of the extracellular domain and construct a prokaryotic expression plasmid.【Method】 The cDNA of duck MHC-Ⅰα chain extracellular domain of mature peptide gene amplified with RT-PCR from healthy duck spleen tissue was cloned into pGEM-T Easy vector by T/A Ligation.After double digestion,MHC-Ⅰα chain extracellular domain of mature peptide cDNA fragment was subcloned into pET-28a (+) vector to construct a prokaryotic expression plasmid.Restriction endonucleases analysis and sequencing were used to conform the recombinant plasmid.【Result】 The duck MHC-Ⅰα chainmature peptide gene of the extracellular domain is 813 bp in length encoding 200 amino acids.The gene shared 89.4%-91.7% nucleotide homology and 78.2%-83.7% amino acid homology with other duck MHC-Ⅰα chain mature peptide gene of the extracellular domain.Phylogenetic tree analysis indicated that the duck MHC-Ⅰα chain mature peptide gene sequence variation was related to genus,the nearer relationship,the higher homology.The duck MHC-Ⅰα chain mature peptide gene of the extracellular domain was correctly inserted into prokaryotic expression vector.【Conclusion】 The result showed that the recombinant plasmid pET/Du MHC-Ⅰ was constructed correctly,which paved the way for further studies on the MHC-Ⅰα protein expression and its biological activities. |
| Key words: duck MHC-Ⅰα chain mature peptide of the extracellular domain prokaryotic expression vector |
