日本血吸虫信号蛋白14-3-3在毕赤酵母菌中的分泌表达及其抗原性分析  

Secreted Expression of Signaling Protein 14-3-3 of Schistosoma japanicom in Pichia pastoris System with Primary Evaluation on its Antigenicity

作　　者：郑美娟[1] 李敏[2] 王志成[2] 罗飞[2] 罗庆礼[2] 储德勇[2] 李丛磊[2] 沈继龙[2] 

机构地区：[1]蚌埠医学院生物化学与分子生物学实验室,蚌埠233003 [2]安徽医科大学病原生物学教研室教育部与安徽省共建重要遗传病基因资源利用重点实验室(安徽医科大学),合肥230032

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第1期12-16,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30571631);国家863高技术研究发展计划(No.2004AA2Z3570)~~

摘　　要：目的在毕赤酵母菌(Pichia pastoris)表达系统中表达日本血吸虫信号蛋白14-3-3(Sj14-3-3),并与原核表达rSj14-3-3比较其抗原性。方法以重组质粒pET28a-rSj14-3-3为模板,PCR扩增Sj14-3-3基因,将特异片段连接到pMD18-T载体,DNA序列分析后,亚克隆目的片段Sj14-3-3至酵母菌分泌表达载体pPICZαB。测序正确后,重组质粒经电转化转染至毕赤酵母菌X-33菌株,经抗生素Zeocin筛选得到高拷贝转化子。经甲醇诱导表达,取诱导上清进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)分析。用间接ELISA法比较毕赤酵母菌表达的rSj14-3-3和原核表达rSj14-3-3检测血吸虫病患者血清抗体的特异性和敏感性。结果目的基因已在酵母菌基因组中得到整合,PCR扩增得到约1 300 bp的片段。经甲醇诱导,Sj14-3-3表达并分泌到培养上清中。表达产物经SDS-PAGE测定为Mr 35 000。Western blotting结果显示,Mr 35 000蛋白可被Sj14-3-3单克隆抗体识别,表明该真核表达产物具有免疫反应性。间接ELISA检测结果表明,该重组蛋白检测36份急性血吸虫病患者血清rSj14-3-3抗体,阳性率为81%。与12份华支睪吸虫感染者血清未见交叉反应,32份健康人血清假阳性反应率为9.3%。以原核表达的rSj14-3-3为抗原,间接ELISA检测,36份急性血吸虫病患者血清的rSj14-3-3,抗体阳性率为88.9%;与12份华支睪吸虫感染者的交叉反应率为16.7%,32份健康人血清假阳性反应率为12.5%,其差异均无统计学意义(P>0.05)。结论在毕赤酵母菌中成功表达了Sj14-3-3,培养上清中产物丰度较高,且免疫反应性良好。Objective To express signaling protein Sj14-3-3 in Pichia pastoris and compare its antigenicity with prokaryotic expression one. Methods Sj14-3-3 gene was amplified from pET28a-Sj14-3-3 recombinant plasmid, cloned into vector pMD18-T followed by sequencing. The Sj14-3-3 gene was subcloned into the expression vector pPICZα-B and transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by sequencing. Three transformants with high copies were obtained when selected under zeocin, and expression was induced with methanol. The culture supematant was collected and tested by SDS-PAGE and Westem blotting. The specificity and sensitivity of eukaryotic expression rSj14-3-3 in Pichia pastoris were compared with that from prokaryotic expression by detecting sera of patients with schistsomiasis by indirect ELISA. Results The Sj14-3-3 gene was integrated into Pichia pastoris, and the gene of interest detected by PCR was with 1 300 bp. After induction by methanol, the Sj14-3-3 gene was expressed and secreted into the medium. The molecular weight of the recombinant protein was determined as about Mr 35 000 by SDS-PAGE. Westem blotting showed that the protein has a high specificity against mouse-anti-Sj14-3-3 monoclonal antibody. The recombinant protein had a promising immune reactivity. Indirect ELISA showed that by using eukaryotic expression rSj14-3-3 in Pichia pastoris, the positive rate in 36 cases of acute schistosomiasis was 81%, with no cross-reactivity in 12 cases of Clonorchis sinensis, 9.3% cross-reactivity in 32 cases of normal sera. While using prokaryotic expression rSj14-3-3 in E.coli, the positive rate in 36 cases of acute schistosomiasis was 88.9%, with 16.7% cross-reactivity in 12 cases of Clonorchis sinensis, 12.5% cross-reactivity in 32 cases of normal sera. There was no statistically significant difference of the results（P〉0.05）. Conclusion The recombinant protein Sj14-3-3 of eukaryotic expression in Pichia pastoris has been successfully harvested and shows a promising

关 键 词：日本血吸虫 信号蛋白14-3-3 毕赤酵母菌 真核表达 

分 类 号：R383.24[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]