青鱼γ-干扰素基因的克隆与表达分析

Clone and expression of interferon gamma(IFN-γ)in black carp,Mylopharyngodon piceus

【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-γcDNA全长为895bp,其开放阅读框大小为549bp,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。

【Objective】This study cloned black carp（Mylopharyngodon piceus）IFN-γgene and analyzed its structural features and expression profiles.【Method】Using cDNA RACE,the 5′and 3′terminal sequences of IFN-γgene cDNA were obtained from black carp.Then,the specific primers were designed and the full length of IFN-γcDNA of black carp was cloned.The nucleic acid sequence and its coded amino acid sequence were analyzed by bioinformatics,homology comparison,phylogenetic tree,and expressionprofiles.Finally,the eukaryotic expression vector of the IFN-γgene was transfected into black carp fin cells for expression in vitro.【Result】The IFN-γcDNA sequence consisted of 549 bp,encoding aputative protein of 182 amino acids.This IFN-γwas 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-γcontained 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-γincreased 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-γcDNA was successfully cloned.The expression of IFN-γ was significantly increased in kidney after poly I：C stimulation and IFN-γgene was expressed in vitro.