曼氏迭宫绦虫翻译控制肿瘤蛋白(TCTP)的真核表达、虫体组织和亚细胞定位及免疫保护效果评价

Eukaryotic expression of a translationally controlled tumor protein(TCTP) fromSpirometra mansoni,assessment of worm tissue,subcellular localization of that protein,and assessment of the protective immunity of recombinant TCTP

目的了解曼氏迭宫绦虫翻译控制肿瘤蛋白(SmTCTP)的虫体组织定位,并以Vero细胞为代替模型观察其亚细胞定位,观察重组抗原在曼氏裂头蚴感染的小鼠中的免疫保护效果。方法构建pEGF-N1-SmTCTP真核表达质粒并转染Vero细胞,利用激光共聚焦荧光显微镜观察SmTCTP重组蛋白在Vero细胞中的分布及亚细胞定位;采用免疫组化法观察SmTCTP在曼氏迭宫绦虫成虫组织中的分布。利用重组抗原做动物免疫保护试验,免疫组小鼠注射重组SmTCTP与等体积完全佐剂混合物,空质粒对照组小鼠注射同重组SmTCTP蛋白等量的GST蛋白与等体积完全佐剂混合物,PBS对照组注射相同体积的PBS溶液。每组动物均免疫3次,采用腹腔内部注射,每次免疫注射量为100μl/只,抗原剂量为50μg/只。第3次免疫后的第2周,所有小鼠均经灌胃感染曼氏裂头蚴5条/只,感染后第6周处死并解剖,收集曼氏裂头蚴并计数,计算曼氏裂头蚴的减虫率。结果双酶切和DNA测序鉴定真核表达重组质粒pEGFN1-SmTCTP构建成功,激光共聚焦观察细胞质与细胞核中均有重组SmTCTP分布,但主要分布在细胞核;免疫组化检测SmTCTP主要分布于曼氏迭宫绦虫成虫的子宫、睾丸和卵黄腺处。动物实验显示,攻虫感染6周的重组质粒SmTCTP免疫小鼠检虫数为(1.00±0.816)条,空质粒组为(2.60±0.966)条,PBS对照组为(3.00±0.738)条,且重组SmTCTP免疫小鼠减虫率为66.7%。结论成功构建pEGF-N1-SmTCTP重组质粒,并在Vero细胞中获得了表达。SmTCTP主要定位于曼氏迭宫绦虫成虫的生殖系统,其引发的信号传递可能对曼氏迭宫绦虫生殖系统的发育和功能起调控作用。重组蛋白SmTCTP可诱导小鼠产生抗曼氏迭宫绦虫裂头蚴的免疫保护作用。

Objective To ascertain the distribution of SmTCTP in tissue of adult Spirometra mansoni tapeworms and its subcellular localization in Vero cells. To determine the protective immunity conferred by recombinant SmTCTP in mice infected with Spirometra znansoni. Methods The recombinant plasmid pEGFP-N1-SmTCTP was constructed and transfected into Vero cells. Confocal microscopy was used to verify the subcellular localization of the SmTCTP in Vero cells. Then, tissue localization of SmTCTP in adult S. rnansoni was performed with an immunohistochemical assay. The extent of protective immunity conferred by SmTCTP was preliminarily evaluated in a mouse experiment using the follow- ing protocol. Mice in the immunized group were injected with a mixture of the recombinant protein and an equal volume of complete adjuvant, mice in the blank plasmid control group were injected with a mixture of GST protein and an equal vol- ume of complete adjuvant, and mice in the PBS control group were directly injected with the same volume of PBS. Ani- mals in each group were intraperitoneally immunized 3 times in week 0, 2, and 4. The immunization dose for each animal was 100μ1 and the antigen dose was 50 μg. Two weeks after the third immunization, 5 S. mansoni larvae were intragas- trically administered to each animal. Six weeks after infection, animals were sacrificed and dissected to collect S. mansoni in order to calculate the rate of the decrease in S. mansoni. Results Double enzyme digestion and DNA Sanger sequencing verified that the recombinant plasmid pEGFP-N1 SmTCTP was successfully constructed. Confocal microscopy indicated that green fluorescence was distributed in the cytoplasm and nucleus, though mainly in the nucleus. Imnmnohis tochemical localization indicated that SmTCTP was mainly distributed in the uterus, testes, and vitellaria of the adult S. mar＊soni. Results of the experiment to confer protective immunity to mice indicated that there were 1.00q-0. 816 larva in the immunized group, 2.60~0. 966 larvae in the blank plasmid control group, and 3.00±0. 738 larvae in the PBS control group. In mice in the immunized group, worms decreased 66.7%o in comparison to the count in the PBS control group, and the worm count differed significantly between the two groups （P〈0.01）. Conclusion The recombinant plasmid pEGFP-NI-SmTCTP was successfully constructed and expressed in Vero cells. SmTCTP was located in the reproductive system of adult S. mansoni and may play an important role in the development and function of the reproductive system of S.mansoni by inducing signaling. SmTCTP conferred significant protective immunity from S. mansoni in mice.