细粒棘球绦虫(中国大陆株)诊断抗原P-29基因的表达、纯化及免疫原性初步分析
师志云;李昭宇;卜阳;王娅娜;李宗吉;马锐;赵巍;
宁夏医科大学;宁夏医科大学附属医院;
Expression and purification of gene encoding the diagnostic antigen P-29 of Echinococcus granulosus and the preliminary analysis on its immunogenicity
摘要 目的对细粒棘球绦虫(Echinococcus granulosus,Eg)诊断抗原P-29(diagnostic antigen P-29)重组质粒进行原核表达、纯化,并初步分析重组蛋白的免疫原性。方法从重组质粒EgP-29/pGEM-T中获取诊断抗原P-29基因,亚克隆于表达载体pET-28a构建基因工程菌株,并表达、纯化重组蛋白,经Western-blot、ELISA分析重组蛋白的免疫原性。结果成功构建原核重组表达载体EgP-29/pET-28a/BL21(DE3)plysS,并纯化出浓度较高的重组蛋白。ELISA检测显示,用表达、纯化的重组蛋白免疫小鼠,诱导产生了特异性抗体IgG,Western-blotting鉴定该抗体能识别重组抗原及天然抗原原头蚴。结论重组蛋白具有良好的免疫原性。
关键词 ：
细粒棘球绦虫 ,
诊断抗原P-29基因 ,
蛋白表达 ,
纯化 ,
免疫原性
Abstract ：To construct the recombinant prokaryotic expression vector for gene encoding the diagnostic antigen P-29 of Echinococcus granolosus and to express and purify the recombinant EgP-29 protein(rEgP-29),this gene was obtained from recombinant plasmid pEgP-29/pGEM-T,subcloned to expression vector pET-28a and transformed to E coli BL21(DE3).The rEgP-29 protein was expressed under the induction with IPTG and purified by His-bind protein purification kit.Then.this protein was used as antigen to immunize ICR mice and the induced immune responses were detected by ELISA and Western blotting.It was demonstrated that the recombinant prokaryotic expression vector EgP-29/pET-28a/BL21(DE3)plysS was successfully constructed and rEgP-29 was expressed and purified efficiently and could elicit effective immune responses,in which the specific IgG antibodies could be induced 6 weeks after immunization.The sera of the immunized mice could recognize specifically rEgP-29 as well as the native protein of protoscolex of E.granulosus.These results indicate that rEgP-29 protein possess good immunogenicity.
Key words ：
Echinococcus granulosus
diagnostic antigen P-29 gene
expression
purification
immunogenicity
收稿日期: 2009-11-20
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