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目的 对旋毛虫新生幼虫p460 0 0抗原基因进行原核表达并检测重组抗原的抗原性。 方法 利用聚合酶链反应 (PCR)扩增目的基因 ,克隆到原核表达载体 pET 2 8a ,构建重组表达质粒 ,经酶切及测序鉴定后转化大肠埃希菌BL2 1(DE3 ) ,以异丙基 β D 硫代半乳糖苷诱导表达。十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)、酶联免疫吸附测定 (ELISA)和蛋白质印迹法 (Westernblotting)分析表达产物。 结果 SDS PAGE结果显示表达产物的分子量约为Mr 480 0 0 ,与理论值相符。ELISA和Westernblotting结果表明 ,重组蛋白可被旋毛虫感染的猪血清和兔抗重组蛋白血清识别 ;兔抗重组蛋白血清可识别旋毛虫新生幼虫抗原Mr 460 0 0蛋白。 结论 成功表达了旋毛虫新生幼虫p460 0 0抗原基因 ,重组抗原具有良好的抗原性。
Objective To express the p460 0 antigen gene of Trichinella spiralis newborn larvae and test the antigenicity of the recombinant antigen. Methods The target gene was amplified by polymerase chain reaction (PCR) and cloned into prokaryotic expression vector pET 2 8a. The recombinant plasmid was constructed and identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21 (DE3) β D thiogalactoside induced expression. The expression products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), enzyme-linked immunosorbent assay (ELISA) and Western blotting. Results SDS PAGE results showed that the molecular weight of the expressed product was about Mr 480 0 0, which was consistent with the theoretical value. ELISA and Western blotting results showed that the recombinant protein could be recognized by Trichinella inoculated swine serum and rabbit anti-recombinant protein serum. Rabbit anti-recombinant protein serum could recognize Mr 460 0 0 protein of Trichinella spiralis newborn larvae. Conclusion The p460 0 0 antigen gene of Trichinella spiralis larvae was successfully expressed, and the recombinant antigen has good antigenicity.