[Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus].

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.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.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.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.［摘要］目的 对猪囊尾蚴排泄分泌抗原中差异表达蛋白富含亮氨酸重复序列结构域15 (leucine-rich repeat containing 15, LRRC15) 进行真核表达, 并预测其抗原表位。方法 通过在线软件ExPASy-PortParam和Protean软件预测LRRC15蛋白分子质量、稳定性、氨基酸序列组成及等电点和T淋巴细胞抗原表位。采用基于PCR的精确合成 (PCR-based accuratesynthesis, PAS) 技术设计全长拼接引物, 合成LRRC15 基因。构建重组质粒pcDNA3.4-LRRC15并转染至人胚胎肾细胞HEK293, 表达LRRC15蛋白, 并进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳 (sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, SDS-PAGE) 和免疫印迹试验 (Western blotting) 鉴定。结果 成功构建了重组质粒pcDNA3.4-LRRC15, 表达分子质量约70 kDa的LRRC15目的蛋白。经ExPASy-PortParam软件预测, LRRC15属于亲水性蛋白, 由644个氨基酸组成, 分子质量为69.89 kDa, 等电点为5.6; 蛋白分子式为C3073H4942N846O953S28, 不稳定系数为50.3, 为一种不稳定蛋白。采用Protean软件预测发现, LRRC15蛋白位于292 ~ 295、353 ~ 361、521 ~ 526、555 ~ 564位氨基酸区段具有T细胞抗原表位优势, 具有亲水性高、柔韧性好、表面可及性大、抗原性指数高的表位位于122 ~ 131、216 ~ 233、249 ~ 254、333 ~ 343、358 ~ 361、368 ~ 372、384 ~ 386、407 ~ 412、445 ~ 450、469 ~ 481、553 ~ 564、588 ~ 594、607 ~ 617、624 ~ 639位氨基酸区段。将重组质粒pcDNA3.4-LRRC15转染至HEK293细胞后, SDS-PAGE和Western blotting分析发现, 在细胞分泌培养基、细胞裂解上清和沉淀中检测到LRRC15蛋白。从细胞培养基中纯化出LRRC15-His融合蛋白, SDS-PAGE显示在分子质量约为70 kDa处出现明显条带, Western blotting能够识别LRRC15重组蛋白条带。结论 成功对猪囊尾蚴排泄分泌抗原中的LRRC15蛋白进行真核表达, 并对其抗原表位进行了生物信息学预测, 为进一步了解该蛋白的生物学功能奠定了基础。.