关键词: 猪囊尾蚴, 排泄分泌抗原, 富含亮氨酸重复序列结构域15, 真核表达, 抗原表位, 生物信息学预测

Abstract: Objective　To conduct eukaryotic expression of the leucine⁃rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope. Methods　The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy⁃PortParam and Protean. The full⁃length splicing primers were designed using PCR⁃based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4⁃LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate⁃polyacrylamide gel electrophoresis (SDS⁃PAGE) and Western blotting. Results　The recombinant pcDNA3.4⁃LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy⁃PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T⁃cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T⁃cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4⁃LRRC15 plasmid into HEK293 cells, SDS⁃PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15⁃His fusion protein was purified from the cell culture medium, and SDS⁃PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein. Conclusions　The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Key words: Taenia solium cysticercus, Excretory secretory antigen, Leucine?rich repeat containing 15, Eukaryotic expression, Antigen epitope, Bioinformatic prediction