摘要
目的克隆弓形虫ROP18基因并构建该基因的原核表达系统。方法根据GenBank发表的弓形虫ROP18序列设计合成1对特异性引物,PCR扩增ROP18抗原基因,并亚克隆到pET-28a原核表达载体,重组质粒转入大肠埃希菌BL21感受态细胞,用IPTG诱导表达,重组蛋白用SDS-PAGE及Western blot鉴定。重组蛋白经纯化后免疫小鼠,ELISA法检测血清抗弓形虫IgG,并通过攻虫试验观察重组蛋白的免疫保护作用。结果成功构建了ROP18基因原核表达质粒pET-28a-ROP18,表达重组蛋白的分子质量单位约为55ku,该蛋白可被鼠抗弓形虫多抗血清识别;经ELISA检测,重组蛋白免疫小鼠产生了较高水平的血清抗体;攻虫试验中,重组蛋白免疫小鼠生存时间较对照组小鼠显著延长(P<0.05)。结论成功构建了ROP18基因原核表达质粒,表达的重组蛋白具有一定的免疫保护作用。
Objective To clone and construct a recombinant expression plasmid for rhoptry protein 18of Toxoplasma gondii in Escherichia coli Methods PCR primers were designed based on the rhoptry protein 18sequence of T.gondii in GenBank.Fragments were amplified using apolymerase chain reaction and then cloned into the vector pET-28a.The recombinant plasmid was transformed into BL21-competent cells of E.coli and induced using IPTG.The expression of the recombinant plasmid was analyzed using SDS-PAGE and Western blotting.Purified ROP18was used to immunize mice and to detect IgG antibodies with ELISA.All mice were challenged with the T.gondii RH strain to test their protective immunity.Results The prokaryotic expression recombinant plasmid pET-28a-ROP18was successfully constructed,and the recombinant protein had a molecular weight of about 55KDa.The protein was recognized by mouse polyclonal antibodies against T.gondii according to Western blotting,which revealed an immune response to some extent.The recombinant protein induced a high titer of anti-ROP18antibodies in immunized mice.Challenge with the T.gondii RH strain revealed significant differences in the survival time for the control group and experimental group;the experimental group had a longer survival time Conclusion A prokaryotic expression recombinant plasmid was successfully constructed.The recombinant protein triggered immuno-protection against a T.gondii challenge in BALB/c mice.
出处
《中国病原生物学杂志》
CSCD
北大核心
2013年第8期708-711,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.30972178,31072127,31001057)
关键词
弓形虫
ROP18
原核表达
Toxoplasma gondii
rhoptry protein 18
prokaryotic expression
