旋毛虫成虫抗原基因的原核表达及表达产物的特性分析

Prokaryotic Expression and Characterization of an Antigenic Gene of Adult Trichinella spiralis

目的 获得旋毛虫成虫抗原基因的原核表达产物并对其进行纯化及免疫特性分析。 方法 旋毛虫成虫Ts87基因片段亚克隆入 PET- 2 8a(+)表达载体 ,异丙基硫代 -β- D-半乳糖苷 (IPTG)诱导表达。亲和层析和电洗脱法纯化表达产物 ,SDS- PAGE、 EL ISA和 Western blotting对表达产物进行分析鉴定 ,并将纯化的表达产物人工免疫兔。结果 SDS- PAGE结果显示 ,表达产物为分子量约为 4 0 k Da的重组蛋白。纯化后免疫兔获得高滴度的抗血清。该基因重组蛋白可被感染旋毛虫的猪、兔血清及人工免疫兔血清所识别 ,与感染囊尾蚴、棘球蚴的患者血清或感染日本血吸虫的兔血清不发生交叉免疫反应。 结论 获得一个新的旋毛虫成虫 Ts87基因重组蛋白 ,该蛋白具有特异的抗原性。

Objective To obtain a purified Ts87 gene expression product of adult Trichinella spiralis and identify its immunogenicity. \ Methods\ Ts87 cDNA was subcloned into PET\|28a(+) expression vector. The transformed bacteria bearing PET\|28a(+)/Ts87 plasmids were induced by IPTG for production of fusion proteins. The expression product \{purified\} with His\|binding affinity chromatography and electro\|elution assay was analyzed by SDS\|PAGE, ELISA and \{Western\} blot, and was used to immunize the rabbits. \ Results \ PET\|28a(+)/Ts87 transformed bacteria \{produced\} the desired fusion protein with a relative molecular weight of 40 kDa. The antisera with high titer were obtained by \{immunizing\} rabbit with Ts87 recombinant protein. Ts87 expression protein was detected as positive reaction with infected \{rabbit\} \{sera\}, infected swine sera and antisera against Ts87 by ELISA. Ts87 protein was also recognized by above\|mentioned sera with Western blotting. However, Ts87 protein failed to react with the patient sera infected with Cysticercus cellulosae or Echinococcus granulosus, and rabbit sera infected with Schistosoma japonicum. \ Conclusion \ A new Trichinella \{recombinant\} protein with specific antigenicity was obtained.