长毛兔和獭兔IFN-γ基因的克隆与真核表达及表达产物的生物学活性  

Cloning and eukaryotic expression of interferon-γ genes of Angola and Rex breeds and biological activity of the expressed products

作　　者：古小彬[1] 杨光友[1] 赖松家[2] 杨厚春 孙家刚[3] 彭文杰 郑晶[4] 廖艳[5] 沈爱琼 王淑贤[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川农业大学动物遗传育种研究所,四川雅安625014 [3]四川省苍溪县畜牧局,四川苍溪628400 [4]四川出入境检验检疫局,四川成都610041 [5]四川省眉山市畜牧局,四川眉山620010

出　　处：《中国兽医科学》2010年第12期1236-1242,共7页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);四川农业大学"双支计划"博士专项项目(00270402)

摘　　要：为扩增长毛兔和獭兔IFN-γ基因及检测其真核表达重组体的生物学活性,采用RT-PCR法从德系安哥拉长毛兔和白色獭兔外周血淋巴细胞总RNA中扩增了IFN-γ基因,测序并序列分析;将该基因亚克隆入pVAX1载体,转染COS-7细胞,MTT法检测其生物学活性。结果表明,长毛兔和獭兔IFN-γ基因大小均为504 bp,两序列间仅在492位存在1个碱基的变异(A→T)。两序列推导的氨基酸序列相同,含167个氨基酸残基。长毛兔和獭兔IFN-γ核苷酸同源性为99.8%,与国内外学者报道的兔IFN-γ核苷酸同源性分别为100.0%和99.8%。基于NJ法构建的进化树发现,国内外学者所报道兔的不同品种均聚类在一起。通过脂质体法转染COS-7细胞,获得具有一定生物学活性的IFN-γ蛋白。结果表明,不同兔品种的IFN-γ基因间差异极小,具有较近的亲缘关系;且pVAX1-IFN-γ转染细胞后能表达具有生物学活性的蛋白。This experiment was conducted to express and analyze the biological activities of interferon-gamma（IFN-γ） of Angola and Rex rabbit breeds.The cDNA of IFN-γ（raIFN-γ） gene was prepared from Con A-stimulated rabbits（Angola and Rex breeds） peripheral blood lymphocyte cells by RT-PCR.The raIFN-γ genes were identified by sequencing and BLAST.Then,the raIFN-γ was cloned into the pVAX1 vector.The recombinant raIFN-γ was transfected into COS-7 cells.In vitro bioactivity of raIFN-γ was determined by a lymphocyte proliferation assay.Sequence analysis showed that raIFN-γ had an open reading frame of 504 base pairs encoding a protein with 167 amino acids.There was only a mutation position（A→T at No.492） in the nucleotide sequences in raIFN-γ between breed Angola and Rex.The IFN-γ gene of Angola breed shared 99.8%nucleotide sequence identity with that of Rex breed.The IFN-γ genes of Angola and Rex breeds shared 100.0% nucleotide sequence identity with other rabbit species in GenBank,respectively.The phylogenetic tree constructed using the NJ method based on genes IFN-γ indicated that the IFN-γ genes of different rabbit breeds available in GenBank were clustered in the same clade.The results suggested that sequence variations of IFN-γ genes were very infinitesimal among different rabbit breeds,and these rabbit breeds had close relationship in genetics.The recombinant plasmid pVAX1-IFN-γ could express the protein with the biological activity after being transfected into COS-7 cells.

关 键 词：长毛兔 獭兔 IFN-Γ 序列分析 生物学活性 

分 类 号：Q511[生物学—生物化学] Q786