| 摘要: |
| 【目的】开展青鱼γ-干扰素(IFN-γ)基因的克隆、结构特征与表达谱分析研究。【方法】采用cDNA末端快速扩增技术获得了青鱼（Mylopharyngodon piceus）IFN-γ基因cDNA的5′和3′末端序列，然后根据其末端序列设计特异性引物，扩增得到青鱼IFN-γ基因cDNA全长序列，对其序列和编码氨基酸序列进行生物信息学、同源性比较及系统进化树分析，同时对其在青鱼各组织中的表达进行分析。通过构建真核表达载体，将其转染青鱼鳍条组织细胞进行表达分析。【结果】青鱼IFN-γ cDNA全长为895 bp，其开放阅读框大小为549 bp，编码182个氨基酸。氨基酸序列比对分析结果显示，青鱼IFN-γ与人、鸡、鼠IFN-γ的序列相似性仅为1.5%～7.7%，与其他鱼类IFN-γ序列相似性较高，为16.3%～92.9%。青鱼IFN-γ分子N末端包含1个信号肽序列、C末端有IFN-γ特征序列（［I/V］-QX-［K/Q］-A-X2-E-［L/F］-X2-［I/V］）以及核定位基序（RRRR）。青鱼IFN-γ蛋白二级结构与高等脊椎动物IFN-γ类似，包含7个α螺旋结构。经Poly I:C诱导后发现，IFN-γ在青鱼头肾、肾、脾、皮肤和鳃组织中表达显著上调，最高的为头肾，其次为脾、皮肤、鳃和肾，而在心脏和脑组织中无显著变化。青鱼γ-干扰素真核表达质粒在青鱼鳍条组织细胞中成功表达IFN-γ。【结论】成功克隆了青鱼IFN-γ基因cDNA，经Poly I:C诱导后，该基因在头肾中上调倍数最为显著，青鱼γ 干扰素在体外获得表达。 |
| 关键词: 青鱼 IFN-γ基因 结构特征 表达分析 |
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| 基金项目:国家“大宗淡水鱼产业技术体系建设专项”（CARS-46-11）；浙江省水生生物资源养护与开发技术研究重点实验室开放基金项目（SS201403） |
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| Clone and expression of interferon gamma (IFN-γ) in black carp,Mylopharyngodon piceus |
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ZHOU Yong,FAN Yuding,HUANG Lili,et al
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| Abstract: |
| 【Objective】This study cloned black carp (Mylopharyngodon piceus) IFN-γ gene and analyzed its structural features and expression profiles.【Method】Using cDNA RACE,the 5′ and 3′ terminal sequences of IFN-γ gene cDNA were obtained from black carp.Then,the specific primers were designed and the full length of IFN-γ cDNA of black carp was cloned.The nucleic acid sequence and its coded amino acid sequence were analyzed by bioinformatics,homology comparison,phylogenetic tree,and expression profiles.Finally,the eukaryotic expression vector of the IFN-γ gene was transfected into black carp fin cells for expression in vitro.【Result】The IFN-γ cDNA sequence consisted of 549 bp,encoding a putative protein of 182 amino acids.This IFN-γ was only 1.5%-7.7% similar to its counterparts in higher vertebrates and 16.3%-92.9% similar to those in other teleost fish. Sequence analysis revealed that the black carp IFN-γ contained the typical IFN-γ motif (［I/V］-QX-［K/Q］-A-X2-E-［L/F］-X2-［I/V］) and a conserved nuclear localization site (RRRR) in the C-terminal end and displayed seven alpha helix structures similar to mammalian IFN-γ.The expression of IFN-γ increased in the kidney,spleen,gills,head kidney,and skin after the poly I:C stimulation.The IFN-γ gene was cloned into the pcDNA3.1 (+) eukaryotic expression vector and expressed in black carp fin cells.【Conclusion】The black carp IFN-γ cDNA was successfully cloned.The expression of IFN-γ was significantly increased in kidney after poly I:C stimulation and IFN-γ gene was expressed in vitro. |
| Key words: Mylopharyngodon piceus IFN-γ gene molecular characterization expression profile |
