| 东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达 |
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| 引用本文： | 翟俊琼,吴亚江,代军威,陈安妮,周妞,王晨,杜雪晴,罗满林,陈武.东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达[J].中国畜牧兽医,2021(1). |
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| 作者姓名： | 翟俊琼 吴亚江 代军威 陈安妮 周妞 王晨 杜雪晴 罗满林 陈武 |
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| 作者单位： | 广州动物园;华南农业大学兽医学院 |
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| 基金项目： | 濒危野生动物调查监管与行业规范项目(20190921)。 |
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| 摘 要： | 本试验旨在通过克隆东北虎γ-干扰素(IFN-γ)基因,研究其分子特征并预测蛋白生物学功能,为后续研究干扰素抗病毒活性做前期准备。通过RT-PCR从ConA诱导过的东北虎血淋巴细胞中扩增东北虎IFN-γ基因并测序,应用生物信息学方法进行序列分析。结果表明:东北虎IFN-γ编码区由504个核苷酸组成,共编码167个氨基酸,蛋白相对分子质量为19.59 ku,等电点为9.03,所编码的蛋白为碱性亲水性蛋白,其中前23个氨基酸可能为信号肽,IFN-γ编码蛋白保守结构域为IFN-γ超家族,且存在跨膜结构,其中1-6位氨基酸为胞内区域,7-28位氨基酸为跨膜区域,29-167位氨基酸为胞外区域;IFN-γ编码蛋白二级结构主要以α-螺旋(58.08%)和无规则卷曲(33.53%)为主,存在5个潜在的B细胞抗原表位,3个潜在的N-糖基化位点;分子进化分析显示,东北虎IFN-γ与GenBank上发表的东北虎、非洲狮、金钱豹、美洲狮、猎豹、家猫、加拿大猞猁、野猪等的核苷酸相似性为80.4%~99.8%,氨基酸相似性为70.5%~100%,东北虎IFN-γ与非洲狮、金钱豹亲缘关系最近,美洲狮、猎豹、家猫、猞猁次之,野猪最远。通过合成改造后的东北虎IFN-γ基因,构建能表达IFN-γ蛋白的重组质粒pPIC9K-IFN-γ,将其导入高效表达系统-毕赤酵母中进行诱导表达,经SDS-PAGE分析,表达蛋白的分子量约17.8 ku,与预期大小相符,表明东北虎IFN-γ成功表达。
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| 关 键 词： | 东北虎 γ-干扰素(IFN-γ) 基因克隆 生物信息学 毕赤酵母 |
Cloning and Bioinformatics Analysis of the Siberian Tigerγ-interferon Gene and Its Expression in Pichia pastoris |
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| ZHAI Junqiong,WU Yajiang,DAI Junwei,CHEN Anni,ZHOU Niu,WANG Chen,DU Xueqing,LUO Manlin,CHEN Wu.Cloning and Bioinformatics Analysis of the Siberian Tigerγ-interferon Gene and Its Expression in Pichia pastoris[J].China Animal Husbandry & Veterinary Medicine,2021(1). |
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| Authors: | ZHAI Junqiong WU Yajiang DAI Junwei CHEN Anni ZHOU Niu WANG Chen DU Xueqing LUO Manlin CHEN Wu |
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| Institution: | (Guangzhou Wildlife Research Center of Guangzhou Zoo,Guangzhou 510070,China;Collgeg of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China) |
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| Abstract: | The purpose of this experiment was to clone the Siberian tigerγ-interferon(IFN-γ)gene,studied its molecular characteristics and predicted the biological function of the protein,making preliminary preparations for the subsequent study of the antiviral activity of interferon.The Siberian tiger IFN-γgene was amplified from ConA-induced Siberian tiger blood lymphocytes by RT-PCR and sequenced to obtain the sequence.Bioinformatics was used for sequence analysis.The results showed that IFN-γgene coding region consisted of 504 nucleotides,encoding a total of 167 amino acids.The relative molecular weight of the Siberian tiger IFN-γamino acid was 19.59 ku,the isoelectric point was 9.03.The encoded protein was a basic hydrophilic protein,of which the first 23 amino acids might be signal peptides.The conserved domain of the IFN-γencoded protein was the IFN-γsuperfamily,and there was a transmembrane structure,in which amino acids 1-6 were intracellular regions and amino acids 7-28 were transmembrane regions,amino acids 29-167 were extracellular regions.The secondary structure of IFN-γencoded protein was mainlyα-helix(58.08%)and random coil(33.53%).There were 5 potential B cell epitopes and 3 potential N-glycosylation sites.Sequence analysis showed that the Siberian tiger IFN-γhad 80.4%-99.8%nucleotide similarity to the Siberian tiger,African lion,leopard,puma,cheetah,domestic cat,Canadian lynx and wild boar published on GenBank,and the amino acid similarity was 70.5%-100%,nucleotide genetic evolution analysis showed that Siberian tiger IFN-γhad the closest relationship with African lions and leopards,followed by puma,cheetah,domestic cat and lynx,and wild boars were farthest.By synthesizing the modified Siberian tiger IFN-γgene,a recombinant plasmid pPIC9K-IFN-γcapable of secreting and expressing IFN-γprotein was constructed,which was introduced into a highly efficient expression system-Pichia pastoris to induce expression,and analyzed by SDS-PAGE,the molecular weight of the expressed protein was about 17.8 ku,which was consistent with the expected size,indicating that the Siberian tiger IFN-γwas successfully expressed. |
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| Keywords: | Siberian tiger γ-interferon(IFN-γ) gene cloning bioinformatics Pichia pastoris |
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