摘要
目的对EgRad54基因进行生物信息学分析,并对细粒棘球绦虫不同发育阶段的表达情况进行检测分析。方法克隆EgRad54基因并对编码产物的理化性质、二、三维结构进行预测和分析;采用MEGA5.0软件构建系统进化树并进行分析;采用qRT-PCR检测细粒棘球绦虫不同发育阶段的EgRad54 mRNA的表达水平。结果 EgRad54基因开放阅读框长度为1 714 bp,NCBI blast比对显示其序列与原始序列相似性为100%,DNAMAN比对相似性为95.40%;EgRad54蛋白含有581个氨基酸,相对分子质量为65.520 45×10^3,理论等电点为8.91;在EgRad54蛋白二级结构中,ɑ-螺旋占44.41%,β-折叠占13.25%,β-转角占6.37%,无规则卷曲占35.97%;三级结构选择123i.A模型,GMQE为0.78,QMEAN为-3.51,序列相似度为69.33%,覆盖率为85.20%;进化树显示EgRad54基因与多房棘球蚴亲缘关系较近;以未消化原头蚴组作为对照组,qRT-PCR检测胃蛋白酶消化原头蚴组和囊泡组Rad54基因相对表达量分别为1.126±0.068和1.111±0.073,差异无统计学意义(F=4.034,P>0.05)。结论成功克隆出细粒棘球绦虫原头蚴Rad54基因,其编码EgRad54蛋白二级结构主要为ɑ-螺旋。该基因在细粒棘球绦虫原头蚴及棘球蚴期不同发育阶段均有表达。
Objectives To analyze the EgRad54 gene bioinformatically and to detect and analyze the expression of Echinococcus granulosus at different developmental stages.Methods The EgRad54 gene was cloned;its physicochemical properties and the two-dimensional and three-dimensional structure of its protein were predicted and analyzed.MEGA5.0 software was used to construct and analyze its phylogenetic tree.Levels of expression of EgRad54 mRNA in different developmental stages of E.granulosus were detected using real-time quantitative PCR(qRT-PCR).Results The open reading frame of the EgRad54 gene was 1714 bp in length.NCBI blast alignment indicated that the EgRad54 gene sequence was 100%similar to the original sequence,and DNAMAN indicated it was 95.40%similar.The EgRad54 protein contains 581 amino acids with a relative molecular weight of 65.52045×10^3 and theoretical isoelectric point of 8.91.Alpha helices accounted for 44.41%of the secondary structure of the EgRad54 protein,beta folds accounted for 13.25%,beta angles accounted for 6.37%,and random coils accounted for 35.97%.A 123 i.a model was selected for the protein’s tertiary structure,with a GMQE of 0.78,QMEAN of-3.51,sequence similarity of 69.33%,and coverage rate of 85.20%.The evolutionary tree indicated that the EgRad54 gene was closely related to E.multilocularis.With an undigested metacercariae group as the control group,the relative expression of the Rad54 gene was 1.126±0.068 in the pepsin-digested metacercariae group and 1.111±0.073 in the vesicle group;relative expression did not differ significantly(F=4.034,P>0.05).Conclusion The Rad54 gene from E.granulosus was successfully cloned,and the secondary structure of the EgRad54 protein that it coded for mainly consisted of alpha helices.This gene is expressed in the protoscoleces of E.granulosus and at different developmental stages.
作者
田春艳
卢帅
文丽梅
赵军
李亚芬
高旖
茹则妮萨·阿卜杜拉
王建华
TIAN Chun-yan;LU Shuai;WEN Li-mei;ZHAO Jun;LI Ya-fen;GAO Yi;RuzenisaoAbudula;WANG Jian-hua(College of Pharmaceutical Sciences,Xinjiang Medical University,Urumqi,China 830054;Pharmacy,The First Affiliated Hospital of Xinjiang Medical University;The State Key Laboratory of athogenesis,Prevention,and Treatment of Diseases Prevalent in Central Asia,The First Affiliated Hospital of Xinjiang Medical University;School of Life Sciences,Nanjing Normal University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2019年第11期1255-1260,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81860666,81560607)
省部共建中亚高发病成因与防治国家重点实验室开放课资助项目(No.SKL-HIDCA-2017-11,SKL-HIDCA-2017-Y7)
新疆维吾尔自治区药学会项目(No.YXH201704)
