红嘴鸥TLR7基因克隆与生物信息学分析

Cloning and Bioinformatics Analysis of TLR7 Gene in Larus ridibundus

【目的】克隆野生鸟类红嘴鸥Toll样受体7(TLR7)基因,并对其编码蛋白进行生物信息学分析,为后期TLR7蛋白的抗病毒活性研究做准备。【方法】采用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)扩增红嘴鸥TLR7基因全序列,通过生物信息学软件分析TLR7基因序列、稀有密码子、相似性及其编码蛋白的理化性质、跨膜区域、信号肽、N-糖基化位点、磷酸化位点和亚细胞定位,分别利用SOPMA和SWISS-MODEL软件预测TLR7蛋白的二级结构和三级结构。【结果】试验成功克隆红嘴鸥TLR7基因(GenBank登录号:MZ668652),全长1720 bp,开放阅读框(ORF)大小为1182 bp,存在39个稀有密码子,其中含8个连续稀有密码子,编码393个氨基酸;红嘴鸥TLR7基因与GenBank中原鸡、红腹角雉、鹌鹑、绿头鸭、黑天鹅、山雀、白鹭、帝企鹅、红喉潜鸟和巴布亚企鹅的TLR7基因相似性分别为85.7%、84.9%、85.4%、87.0%、87.8%、86.8%、91.0%、93.1%、93.1%和93.1%。系统进化树结果显示,其与白鹭的亲缘关系最近。TLR7蛋白分子式为C_(2080)H_(3256)N_(556)O_(567)S_(19),分子质量约为63 ku,理论等电点为9.25;该蛋白不存在跨膜结构和信号肽,含有6个N-糖基化位点和33个磷酸化位点,是疏水性蛋白,主要存在于细胞质中;TLR7蛋白二级结构主要以α-螺旋为主,约占48.09%,其次为无规则卷曲(32.06%)、延伸链(13.23%)和β-转角(6.62%),TLR7蛋白的三级结构与二级结构一致,且与模型蛋白人TLR7蛋白相似性为68.78%。【结论】红嘴鸥TLR7基因与白鹭进化关系较近,含有8个连续稀有密码子,体外表达较难,研究为进一步探索红嘴鸥TLR7蛋白抗病毒免疫机制提供重要参考。

【Objective】The Toll-like receptor 7(TLR7)gene of Larus ridibundus was cloned,and its coding protein was analyzed by bioinformatics,so as to prepare for the study of antiviral activity of TLR7 protein in the later stage.【Method】The whole TLR7 gene sequence of Larus ridibundus was amplified by rapid amplification of cDNA ends(RACE).The TLR7 gene sequence,rare codon,similarity and physical and chemical properties,transmembrane region,signal peptide,N-glycosylation site,phosphorylation site and subcellular localization of TLR7 protein were analyzed by bioinformatics software.The secondary structure and tertiary structure of TLR7 protein were predicted by SOPMA and SWISS-MODEL softwares,respectively.【Result】The TLR7 gene of Larus ridibundus(GenBank accession No.:MZ668652)was successfully cloned.The total length of the gene sequence was 1720 bp,the size of open reading frame(ORF)was 1182 bp,and there were 39 rare codons,including 8 consecutive rare codons,encoding 393 amino acids.The similarity of TLR7 gene between Larus ridibundus and Gallus gallus,Tragopan temminckii,Coturnix coturnix,Anas platyrhynchos,Cygnus atratus,Paridae,Egretta garzetta,Aptenodytes forsteri,Gavia stellata,Pygoscelis papua in GenBank was 85.7%,84.9%,85.4%,87.0%,87.8%,86.8%,91.0%,93.1%,93.1%and 93.1%,respectively.The phylogenetic tree showed that it had the closest genetic relationship with Egretta garzetta.The molecular formula was C_(2080)H_(3256)N_(556)O_(567)S_(19),the molecular weight of TLR7 protein was about 63 ku.The theoretical isoeletric point was 9.25.TLR7 protein had no transmembrane structure and signal peptide.It contained 6 N-glycosylation sites and 33 phosphorylation sites.It was a hydrophobic protein and mainly exists in the cytoplasm.The secondary structure of TLR7 protein was mainly alpha helix dominated,accounting for about 48.09%,followed by random coil(32.06%),extended chain(13.23%)and beta turn(6.62%).The tertiary structure of TLR7 protein was consistent with the secondary structure,and the similarity with the model protein human TLR7 protein was 68.78%.【Conclusion】The TLR7 gene of Larus ridibundus was closely related to the evolution of Egretta garzetta,it contained 8 consecutive rare codons and was difficult to express in vitro.The study provided an important reference for further exploring the antiviral immune mechanism of TLR7 protein in Larus ridibundus.