Eukaryotic expression of calreticulin of Echinococcus multilocularis (EmCRT) and prediction of T- and B-cell epitopes of EmCRT.
Eukaryotic expression of calreticulin of Echinococcus multilocularis (EmCRT) and prediction of T- and B-cell epitopes of EmCRT.
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DOI：
10.13350/j.cjpb.201207
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发表时间：
2020-01-01
期刊：
影响因子：
--
通讯作者：
Zhao, Li-Mei
中科院分区：
文献类型：
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作者：
Chen, Lu-Juan;Cheng, Zhe;Zhao, Li-Mei
目的：构建多房棘球蚴钙网蛋白（EmCRT）真核表达载体，鉴定其在HeLa细胞中的表达，并对该蛋白的T细胞和B细胞表位进行生物信息学分析。方法：根据大肠杆菌钙网蛋白基因序列设计特异性引物。多房的用PCR方法从大肠杆菌中扩增EmCRT基因。多室原孢菌为模板。将PCR产物插入真核表达载体pcDNA3.3-HA，构建重组质粒pcDNA3.3-HA-EmCRT。经PCR、酶切、鉴定和测序后，用脂质体转染试剂将重组质粒pcDNA3.3-HA-EmCRT瞬时转染Hela细胞。采用免疫印迹和免疫荧光法检测EmCRT在Hela细胞中的表达。利用ProtParam软件预测EmCRT的理化性质，并利用SignalP 4.1 Server软件预测EmCRT的信号肽序列。使用PSORT II预测来预测亚细胞位置。使用TMHMM 2.0预测跨膜结构域。分别用SOPMA和SWISS-MODEL对蛋白质的二级和三级结构进行了预测。使用DNAStar软件分析B细胞表位，该软件用于分析蛋白质的亲水性、柔性、抗原性和表面可及性。使用SYFPEUTHI分析T细胞表位。结果成功构建了EmCRT真核表达载体，Western blotting和免疫荧光检测显示EmCRT在Hela细胞中得到高效表达。预测EmCRT蛋白的分子量为45.44*10~3，其等电点为4.47。该蛋白含有信号肽序列和跨膜结构域，可能定位于细胞质中。预测EmCRT蛋白具有6个组合的T细胞和B细胞表位，其位于氨基酸51-88、112-154、149-178、185-198、244-263和280-309。结论：EmCRT的表达及其T、B细胞表位的预测为后续开发高效的E.多房的
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.