摘要
目的克隆、表达细粒棘球蚴分离株Eg10基因,并研究其重组蛋白的免疫特性。方法将细粒棘球蚴Eg10基因亚克隆于表达载体pET28a,转化重组质粒至大肠埃希菌BL21进行融合表达,His-bind树脂纯化系统纯化重组融合蛋白,以之作为抗原免疫小鼠。48只ICR小鼠随机均分为4组,A、B组为对照组,分别注射不含抗原的磷酸盐缓冲液(PBS)和福氏佐剂+PBS,每鼠皮下注射100μl。C、D组为免疫组,注射用福氏佐剂乳化的重组抗原Eg10,抗原量分别为0.1mg/μl和0.5mg/μl,每鼠皮下注射100μl。各组小鼠每隔2周免疫1次,共免疫3次。于免疫前和免疫后2、4、6、8和10周采血以获得抗血清。通过蛋白质印迹(Westernblotting)分析和ELISA检测重组抗原的免疫学特性。结果成功构建含目的片段Eg10的基因工程菌株。Westernblotting分析结果表明,免疫血清能识别重组抗原Eg10。ELISA检测结果显示,用重组蛋白免疫小鼠可诱导产生特异性抗体。统计学分析表明,免疫后C、D组抗体水平逐渐升高,至第8周抗体滴度达最高值,分别为(1.143±0.253)和(1.254±0.070);A、B组抗体一直维持在较低水平。第2、4、6、8和10周,A、B与C、D组间抗体水平差异有统计学意义(均P<0.05),C、D组间差异无统计学意义(P>0.05)。结论成功表达细粒棘球蚴重组蛋白Eg10,该蛋白有一定的免疫原性。
Objective To clone and express Eg10 gene of Echinococcus granulosus, and investigate the immunological characteristic of the recombinant. Methods Eg10 gene was subcloned into pET28a vector. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. The recombinant protein was purified with His-bind purification kit. Forty-eight mice were randomly divided into 4 groups. Mice in groups A and B were injected with PBS and PBS＋Freund′s adjuvant（100 μl） as control. Mice in groups C and D were immunized with 10 mg and 50 μg purified Eg10 antigen formulated in Freund′s adjuvant, respectively. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization. The imm- unological characteristics of recombinant Eg10 was analyzed by Western blotting and ELISA. Results The recombinant Eg10 protein （Mr 31 000） was expressed in E. coli BL21. The recombinant Eg10 and expression product of PET28a/Eg10 were recognized by sera from mice immunized with recombinant Eg10. ELISA showed that the titer of IgG reached a peak at the 8th week in groups C and D, the level of IgG in sera of groups C or D was higher than that of groups A or B （P 0.05） at the 2nd, 4th, 6th, 8th, and 10th week. There was no significant difference in the level of IgG between group C and group D （P0.05）. Conclusion The Eg10 gene has been expressed with immunogenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2010年第5期339-342,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金项目(No30260105)
宁夏自然科学基金(NoNZ0540)~~
