重组杜氏利什曼原虫延伸因子1-α与β-微管蛋白诱导BALB/c小鼠Th2型免疫应答的研究  

Recombinant elongation factor 1-α and β-tubulin of Leishmania donovani induce the Th2 immune response in BALB/c mice

作　　者：刘鹏[1] 姚梦 杨健[1] 敬保迁[1] LIU Peng;YAO Meng;YANG Jian;JING Bao-qian(Institute of Immunology and Molecular Biology,North Sichuan Medical College,Nanchong 637000,Sichuan,China;The Second College of Clinical Medicine,North Sichuan Medical College)

机构地区：[1]川北医学院免疫学与分子生物学研究所,四川南充637000 [2]川北医学院第二临床医学院

出　　处：《中国病原生物学杂志》2018年第6期592-596,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30872213);川北医学院校级课题(No.75401013)

摘　　要：目的评价重组杜氏利什曼原虫延伸因子1-α(recombinant Elongation factor 1-α,rEF 1-α)和β-微管蛋白(recombinantβ-tubulin,rβ-tubulin)的免疫刺激效应及诱导免疫应答类型。方法从杜氏利什曼原虫四川人分离株基因组中扩增EF 1-α、β-tubulin基因,以分子克隆技术构建pQE30-EF 1-α、pQE30-β-tubulin重组质粒。将酶切与测序鉴定正确的重组子转化E.coil M15菌株,用IPTG诱导表达rEF 1-α和rβ-tubulin,表达产物经镍柱亲和层析纯化后辅以弗氏佐剂制成疫苗免疫BALB/c小鼠,采用Western blot法检测小鼠抗血清的特异性,ELISA法检测抗血清效价。分离各组小鼠脾细胞,体外培养60h后再次接受相同抗原刺激,检测IL-4、IL-12、IFN-γ水平。结果重组菌表达的rEF 1-α、rβ-tubulin均以包涵体形式存在,两表达产物免疫小鼠后获得的抗血清均具有特异性,效价分别为1∶800和1∶1 600。rEF 1-α、rβ-tubulin免疫小鼠脾细胞培养上清IL-4含量均高于对照组(P<0.05),IL-12、IFN-γ与对照组比较差异无统计学意义(P>0.05)。结论成功表达了杜氏利什曼原虫rEF 1-α、rβ-tubulin,两种重组蛋白均可诱导Th2型免疫应答。Objective To evaluate stimulation of an immune response with recombinant elongation factor 1-α（EF 1-α）andβ-tubulin of Leishmania donovani and the type of immune response induced. Methods The EF 1-αandβ-tubulin genes were amplified from the genomic DNA of L.donovani and cloned into a pQE30 plasmid vector.The recombinant plasmids were verified as correct with enzyme digestion and sequencing and were transformed into E.coli M15.Plasmid expression was induced with IPTG,and the recombinant proteins were purified using Ni-chelating affinity chromatography.The recombinant proteins were combined with Freund’s adjuvant and used as vaccines to immunize BALB/c mice.The specificity of mouse antiserum was determined using Western blotting and the antiserum titer was determined using ELISA.Spleen cells were isolated from each group of mice and cultured for 60 hin vitro to detect levels of IL-4,IL-12,and IFN-γafter stimulation with the same antigen. Results rEF 1-αand rβ-tubulin were expressed in the form of inclusion bodies.After mice were immunized with the recombinant proteins,the two antisera specifically recognized rEF 1-αand rβ-tubulin.The titer of anti-rEF 1-αantibodies in antiserum was 1：800 and that of anti-rβ-tubulin antibodies was 1：1 600.The level of IL-4 in the supernatant of cultured spleen cells from mice immunized with rEF 1-αand rβ-tubulin was higher than that in the control group treated with PBS.Levels of IL-12 and IFN-γdid not differ from those in the control group treated with PBS. Conclusion The EF 1-αandβ-tubulin genes of L.donovani were cloned and successfully expressed.Both recombinant proteins induced a Th2 immune response in BALB/c mice.

关 键 词：杜氏利什曼原虫 重组蛋白 Th2型免疫应答 

分 类 号：R382.22[医药卫生—医学寄生虫学]