| 摘要: |
| [目的]筛选新的高效抗日本血吸虫病疫苗.[方法]运用“电子cDNA文库”筛选法和快速cDNA末端扩增技术,对日本血吸虫新基因Sj314C10进行了克隆,构建了原核表达载体pET-28a(+)-Sj314C10,并探索了各种条件对表达蛋白的影响;用生物学软件初步预测了该基因的编码蛋白,用亲和层析法对表达产物进行纯化并完成了表达产物的抗原性检测及其对动物的保护性研究.[结果]表达产物约50 ku,为包涵体蛋白,温度、IPTG浓度和表达时相的改变对该包涵体蛋白的可溶性表达无显著影响;生物信息学分析表明,SJ314C10编码蛋白与杆状病毒凋亡抑制蛋白的重复结构域有78%的同源性,纯化蛋白抗原性良好;小鼠抗血吸虫尾蚴攻击试验显示,与佐剂对照组相比,蛋白免疫组获得了部分保护作用,这为血吸虫候选分子疫苗的研究增添了新内容.[结论]日本血吸虫Sj314C10基因得到了成功表达,所表达蛋白具有良好的抗原性,对小鼠具有一定的免疫保护作用. |
| 关键词: 日本血吸虫 新基因Sj314C10 表达纯化 功能分析 |
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| 基金项目:国家“863”计划项目(2001AA215151)；国家血吸虫病防治研究专项（2004AA223510）；上海市重大科技攻关项目(03D219231) |
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| Expression and Functional Analysis of Sj314C10,a Novel Gene of Schistosoma japonicum |
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| Abstract: |
| 【Objective】 The study is to screen novel and high-effective vaccines against schistosomiasis.【Method】 A novel S.japonicum gene Sj314C10 was cloned into a prokaryotic expression vector pET28a(+) and induced expression in different conditions.A 50 ku insoluble inclusion body fusion protein was expressed in E.coli BL21 and purified by His Band affinity chromatography.【Result】 The analyses of the structure and functional motifs of Sj314C10 recombinant protein showed that the amino acid sequence of the protein had 78% homology with the Baculovirus IAP repeat domain.The changes of culture temperature,concentrations of IPTG,different expression time had no significant influence on the dissoluble characteristics of the inclusion body protein.Purified recombinant protein had good antigenicity.Immune experiments in mice showed that the purified recombinant protein induced partial protection against Schistosoma japonicum cercaria challenge compared with the adjuvant control.The results added new material for the research of molecular vaccines against schistosomiasis.【Conclusion】 Sj314C10 gene of S.japonicum was successfully expressed.The expressed protein had good antigenicity and certain immunoprotective effect to mice. |
| Key words: Schistosoma japonicum novel gene Sj314C10 expression and purification function analysis |
