摘要
目的构建多房棘球绦虫钙网蛋白(EmCRT)的真核表达载体,鉴定重组EmCRT蛋白在Hela细胞中的表达,并对其进行T、B细胞表位预测等生物信息学分析。方法根据多房棘球绦虫钙网蛋白基因序列设计特异引物,以多房棘球绦虫原头蚴cDNA为模板,PCR扩增EmCRT基因片段。将此片段插入真核表达载体pcDNA3.3-HA,构建重组质粒pcDNA3.3-HA-EmCRT,并将PCR、酶切和测序鉴定正确的质粒转染Hela细胞,应用Western blot和细胞免疫荧光法检测重组EmCRT蛋白在细胞中的表达。利用ProtParam预测EmCRT的理化性质,SignalP 4.1 Server预测其信号肽序列,PSORT II Prediction预测其亚细胞定位,TMHMM 2.0预测其跨膜结构域,SOPMA和SWISS-MODEL预测其二、三级结构,DNAStar软件分析其亲水性、柔韧性、抗原指数以及表面可及性,并推测可能的B细胞表位;采用SYFPEUTHI的T细胞表位预测工具分别预测细胞毒性T细胞(CTL)和辅助T细胞(Th)表位。结果成功构建了EmCRT的真核表达载体,经Western blot和细胞免疫荧光检测EmCRT在Hela细胞中高效表达。EmCRT蛋白的相对分子质量为45.44×10^(3),等电点为4.47,预测该蛋白含有1个信号肽序列和1个跨膜区域,可能定位于细胞质,具有6个T、B细胞联合表位,分别为51-88 aa、112-154 aa、149-178 aa、185-198 aa、244-263 aa、280-309 aa。结论真核表达的重组多房棘球绦虫钙网蛋白相对分子质量为45.44×10^(3),预测含有T、B细胞表位,为高效抗多房棘球绦虫表位疫苗的研发奠定了基础。
Objectives To construct a vector for eukaryotic expression of Echinococcus multilocularis calreticulin(EmCRT)and to identify its expression in Hela cells,and to bioinformatically analyze the T-and B-cell epitopes of this protein.Methods Specific primers were designed based on the calreticulin gene sequence of E.multilocularis.The EmCRT gene was amplified using PCR with cDNA from E.multilocularis protoscoleces as a template.The PCR products were then inserted into the eukaryotic expression vector pcDNA3.3-HA to construct the recombinant plasmid pcDNA3.3-HA-EmCRT.After PCR,cleavage with restriction enzymes,identification and sequencing,the recombinant plasmid pcDNA3.3-HA-EmCRT was transiently transfected into Hela cells with liposome transfection reagents.Expression of EmCRT in Hela cells was detected using Western blotting and immunofluorescence.The physical and chemical properties of EmCRT were predicted using ProtParam,and its signal peptide sequences were predicted using SignalP 4.1 Server.Subcellular location was predicted using PSORT II Prediction.Transmembrane domains were predicted using TMHMM 2.0.The protein’s secondary and tertiary structures were predicted using SOPMA and SWISS-MODEL respectively.B-cell epitopes were analyzed using the software DNAStar,which was used to analyze the protein’s hydrophilicity,flexibility,antigenicity,and surface accessibility.T-cell epitopes were analyzed using SYFPEUTHI.Results The eukaryotic expression vector EmCRT was successfully constructed,and EmCRT was highly expressed in Hela cells according to Western blotting and immunofluorescence.The molecular weight of the EmCRT protein was predicted to be 45.44×10^(3),and its isoelectric point was 4.47.The protein contained a signal peptide sequence and a transmembrane domain and might be located in the cytoplasm.EmCRT protein was predicted to have 6 combined T-and B-cell epitopes that were located at animo acids 51-88,112-154,149-178,185-198,244-263,and 280-309.Conclusion The expression of EmCRT and prediction of its T-and B-cell epitopes have provided a theoretical basis for the subsequent development of a highly effective epitope-based vaccine against E.multilocularis.
作者
陈路娟
程喆
王彦海
赵利美
CHEN Lu-juan;CHENG Zhe;WANG Yan-hai;ZHAO Li-mei(Department of Pathogen Biology,School of Preclinical and Forensic Medicine,Baotou Medical College,Baotou,Inner Mongolia,China 014060;Parasitology Research Laboratory,School of Life Sciences,Xiamen University,Xiamen,Fujian,China 361102)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第12期1397-1403,共7页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81860362)
内蒙古自治区自然科学基金项目(No.2018BS08013)
内蒙古自治区高等学校科学研究项目(No.NJZZ18185)
包头医学院博士科研启动基金项目(No.BSJJ201805)。
关键词
多房棘球绦虫
钙网蛋白
真核表达载体
抗原表位预测
Echinococcus multilocularis
calreticulin
eukaryotic expression vector
epitope prediction
