摘要
本研究旨在克隆犬乳腺组织中犬乳铁蛋白(LTF)基因CDS序列并进行详尽的生物信息学分析,为下一步的研究奠定基础。从犬乳腺组织中扩增得到LTF CDS全长,并克隆至pGEX-4T-1原核表达载体,利用双酶切与测序验证重组质粒pGEX-4T-LTF,最后使用生物信息学软件对LTF进行生物信息学分析。结果显示,成功克隆了全长2127 bp的LTF的CDS;LTF属于可溶性蛋白,分子质量为77 kDa,等电点为8.62;二级结构中α-螺旋(Hh)占36.44%,延长链(Ee)占15.11%,无规则卷曲(Cc)占48.45%;三级结构中无规则卷曲较多,α-螺旋与β-折叠较为集中;跨膜区分析得知LTF蛋白708个氨基酸全部位于细胞膜表面;信号肽预测剪切位点位置在犬LTF基因蛋白的第19-20位;B细胞表位预测表明,有5个潜在的B细胞优势表位。本研究结果可为犬乳铁蛋白生物学功能研究以及开发利用提供详实的数据支持。
The aim of this study was to clone the CDS sequence of lactoferrin (LTF) gene from canine breast tissue,amplify its full length and clone it into prokaryotic expression vector pGEX-4T-1.The recombinant plasmid pGEX-4T-LTF was verified by double endonuclease digestion and sequencing.Finally,bioinformatics software was used to analyze LTF.The results showed that the full length of LTF gene was 2127 bp and the recombinant pGEX-4T-LTF was expressed.The expressed LTF was a soluble protein with the molecular weight of 77 kDa and isoelectric point of 8.62.In the secondary structure of the expressed LTF,α-helix (Hh) accounted for 36.44%,extended strand (Ee) for 15.11% and irregular crimp (Cc) for 48.45%.In the tertiary structure,there were more irregular crimps and concentrated α-helix and β-sheets.Transmembrane region analysis showed that all 708 amino acids of the LTF protein were located on the surface of cell membranes and the predicted splicing site of signal peptide located between the 19 and 20 of the canine LTF gene protein.B-cell epitope prediction showed that there were five potential B-cell dominant epitopes.This study provided essential data for future research on the biological function of canine lactoferrin.
作者
李航
陈宗艳
刘光清
李传峰
LI Hang;CHEN Zongyan;LIU Guangqing;LI Chuanfeng(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)
出处
《中国动物传染病学报》
CAS
北大核心
2021年第6期82-89,共8页
Chinese Journal of Animal Infectious Diseases
基金
国家重点研发计划项目(2016YFD0501003)
上海市科技兴农创新项目(2019No.3-3)。
关键词
犬乳铁蛋白
基因克隆
序列分析
生物信息学分析
Canine lactoferrin
gene cloning
sequence analysis
bioinformatics analysis
