羚牛IFN-γ基因克隆、生物信息学分析与原核表达  

Cloning,Bioinformatics Analysis and ProkaryoticExpression of IFN-γGene in Takin

作　　者：柳丽 姚宇航 刘晨阳 张文涛 马俊杰 昝林森[1,2] 成功[1,2] LIU Li;YAO Yuhang;LIU Chenyang;ZHANG Wentao;MA Junjie;ZAN Linsen;CHENG Gong(College of Animal Science and Technology,Northwest A&F University,Yangling Shaanxi 712100,China;National Beef Cattle Improvement Centre of Northwest A&F University,Yangling Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]西北农林科技大学国家肉牛改良中心,陕西杨凌712100

出　　处：《西北农业学报》2025年第2期319-328,共10页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省林业科技创新计划(SXLK2020-0301,SXLK2021-0102)。

摘　　要：旨在研究羚牛γ干扰素(IFN-γ)基因结构与功能,为进一步增强羚牛的免疫调节能力提供理论依据。通过RT-PCR方法克隆羚牛IFN-γ基因,并运用多种生物信息学分析工具对其结构与功能进行深入研究。将IFN-γ基因序列连入原核表达载体,并转化大肠杆菌诱导表达。结果显示,羚牛IFN-γcDNA序列全长501 bp,编码166个氨基酸。蛋白高级结构预测结果显示,二级结构以α螺旋为主,存在13个磷酸化修饰位点,并含有1个信号肽、1个低度复杂区、1个跨膜结构域及1个IFN-γ结构域,其中IFN-γ结构域位于胞外区。序列比对分析发现其与绵羊、山羊、家牛、水牛、人、黑猩猩、小鼠、鸡的核苷酸序列同源性分别为99.0%、98.8%、97.2%、96.2%、75.8%、75.4%、64.1%、54.1%,氨基酸序列同源性分别为99.4%、99.4%、95.8%、95.8%、62.7%、62.7%、43.9%、33.5%。系统进化树分析结果显示,羚牛与山羊、绵羊的亲缘关系最近。经SDS-PAGE和Western-blot检测发现,重组IFN-γ蛋白以可溶性形式大量表达,分子质量约为34 ku。通过密码子优化及不同浓度IPTG诱导表达等原核表达优化策略发现,密码子优化能促进重组蛋白的高效表达,且最佳诱导浓度为0.9 mmol·L^(-1)。The objective of this study was to explore the structure and function of the interferonγ(IFN-γ)gene in takin,with the aim of providing a theoretical basis for enhancing the immune regulation capacity of this species.In this study,the IFN-γgene of takin was cloned via RT-PCR,a comprehensive analysis of its structure and function were conducted using variety of bioinformatics tools.The IFN-γgene sequence was connected to the prokaryotic expression vector and transformed into E.coli to induce its expression.The results showed that the IFN-γcDNA sequence of takin was 501 bp in length and encoded 166 amino acids.The prediction of the protein’s higher order structure showed that the secondary structure was dominated byαhelix,with 13 phosphorylation modification sites,a signal peptide,a low-grade complex region,a transmembrane domain and an IFN-γdomain.The IFN-γdomain is located in the extracellular region.Sequence alignment analysis showed that the nucleotide sequence homology with sheep,goat,cattle,buffalo,human,chimpanzee,mouse and chicken was 99.0%,98.8%,97.2%,96.2%,75.8%,75.4%,64.1%,and 54.1%,respectively.The amino acid sequence homology was 99.4%,99.4%,95.8%,95.8%,62.7%,62.7%,43.9%,and 33.5%,respectively.The result of the phylogenetic tree analysis also showed that takin was most closely related to goats and sheep.SDS-PAGE and Western-blot showed that the recombinant IFN-γprotein was abundantly expressed in soluble form with a molecular weight of about 34 ku.Through optimization strategies for prokaryotic expression,such as codon optimization and IPTG induction expression at different concentrations,it was found that codon optimization promoted the efficient expression of recombinant proteins,and the optimal induction concentration was determined to be 0.9 mmol·L^(-1).This study lays a foundational understanding for comprehensive functional exploration of IFN-γgene in takin,as well as the potential application of interferonγin takin.These findings provide novel insights into takin conservation effort

关 键 词：羚牛 IFN-Γ 基因克隆 生物信息学分析 原核表达 IPTG诱导 

分 类 号：Q95[生物学—动物学]