牛环形泰勒虫AMA1基因表达载体的构建及生物信息学分析  

Construction of Thelieria annulata AMA1 Gene Expression Vector and Bioinformatics Analysis

作　　者：樊新丽[1] 郑会珍 翟雪洁 范士龙 马英 陈宋琴 韦丽婷 刘一凡 巴音查汗[1] 张杨[1] FAN Xin-li;ZHENG Hui-zhen;ZHAI Xue-jie;FAN Shi-long;MA Ying;CHEN Song-qin;WEI Li-ting;LIU Yi-fan;Bayinchahan;ZHANG Yang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi,Xinjiang,830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《动物医学进展》2021年第12期6-13,共8页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0500401);新疆维吾尔自治区高校科研计划项目(XJEDU2017S014)。

摘　　要：为构建牛环形泰勒虫AMA1(TaAMA1)截短基因原核、真核表达载体,对其编码的蛋白进行生物信息学分析,以牛环形泰勒虫阳性DNA为模板,在牛环形泰勒虫AMA1基因的开放性阅读框内设计特异性引物,采用PCR扩增AMA1基因,构建其原核表达质粒pET28a-AMA1及真核表达质粒pEGFP-AMA1。通过生物信息学在线软件分析TaAMA1基因的系统进化树及该蛋白的理化性质、磷酸化位点、亚细胞定位、抗原表位及二级结构,构建该重组蛋白系统发育进化树。结果显示,TaAMA1基因PCR扩增产物大小为828 bp,与预期片段一致,构建的重组质粒经双酶切鉴定表明目的基因插入正确,测序结果与GenBank已收录的牛环形泰勒虫AMA1基因序列(KX231677.1)同源性为99.72%,同源性及系统进化树分析发现与小泰勒虫氨基酸序列同源性最大为69.9%,生物信息学分析发现TaAMA1基因编码蛋白含276个氨基酸,分子式为C_(1331)H_(2134)N_(398)O_(413)S_(4),分子质量为30.448 ku,理论等电点为9.94,有38个磷酸化修饰位点,无跨膜区及信号肽,亚细胞定位预测位于细胞核的比例为87%;B细胞抗原表位分析发现该蛋白含有9个潜在抗原表位;TaAMA1蛋白的二级结构中α-螺旋、β-转角、延伸链和无规则卷曲和延伸链分别占23.91%、7.61%、49.64%和18.84%。成功构建重组原核表达质粒pET28a-AMA1及真核表达质粒pEGFP-AMA1并进行生物信息学分析,为牛环形泰勒虫AMA1蛋白生物学功能及入侵机制研究奠定了基础。To construct the prokaryotic and eukaryotic expression vectors of the protein encoded by the truncated AMA1(TaAMA1)gene of Theileria annulata,ahnd analyze the bioinformatics,the positive DNA of Theileria annulata was used as a template,primers were designed according to the open reading frame of the AMA1 gene of Theileria annulata,and the AMA1 gene was amplified by PCR to construct its prokaryotic expression plasmid pET28a-AMA1 and eukaryotic expression plasmid pEGFP-AMA1.The phylogenetic tree of TaAMA1 gene and the physical and chemical properties,phosphorylation sites,subcellular localization,and secondary structure of TaAMA1 gene were analyzed through bioinformatics online software.The sequencing result was consistent with the Thelieria annulata AMA1 gene sequence(KX231677.1).The alignment consistency was 99.72%.The homology and phylogenetic tree analysis showed that the amino acid sequence similarity to the Thelieria parva was the greatest.Bioinformatics analysis showed that the TaAMA1 gene encoding protein contains 276 amino acids,the molecular formula was C_(1331)H_(2134)N_(398)O_(413)S_(4),and the molecular mass unit was 30.448 ku,the theoretical isoelectric point was 9.94,there were 38 phosphorylation sites,no transmembrane region and signal peptide,and the proportion of subcellular localization predicted to be located in the nucleus is 87%;in the secondary structure of TaAMA1 protein,α-helix,β-turn,random crimp and extended chain accounted for 23.91%,7.61%,49.64%and 18.84%,respectively.The recombinant prokaryotic expression plasmid pET28a-AMA1 and the eukaryotic expression plasmid pEGFP-AMA1 were successfully constructed and analyzed by bioinformatics.The results laid the foundation for the study of the biological function and invasion mechanism of the AMA1 protein of Thelieria annulata.

关 键 词：牛 环形泰勒虫 AMA1基因 表达载体构建 生物信息学分析 

分 类 号：S852.723[农业科学—基础兽医学] S858.23[农业科学—兽医学]