CAJ | 학술논문

目的选取致病性钩端螺旋体(简称钩体)中高度保守,同时是钩体外膜蛋白中含量最多的两个脂蛋白LipL32和LipL21构建成融合基因DNA疫苗pVAX1/LipL21-LipL32,观察在BALB/c小鼠中重组DNA疫苗诱导免疫应答反应的能力.方法采用连接引物PCR构建融合基因LipL21-LipL32,并将其插入真核表达载体构成重组DNA疫苗pVAX1/LipL21-LipL32,脂质体转染人胚肾细胞(HEK293细胞)后Western Blot验证重组DNA疫苗在真核细胞中的表达,并将其肌注BALB/c小鼠,用显微凝集试验(MAT)检测所产生的特异性抗体与问号钩体的凝集效价,用IL-10和TNF-β细胞因子试剂盒检测体液免疫和细胞免疫应答水平.结果 Western Blot分析显示重组DNA疫苗pVAX1/LipL21-LipL32在HEK293细胞中得到表达,小鼠动物实验结果显示重组DNA疫苗能有效地诱导机体的体液免疫和细胞免疫应答.结论成功构建钩体融合基因LipL21-LipL32重组DNA疫苗,所表达的融合蛋白能诱导特异的免疫应答反应,为进一步研究和发展新型的钩体病疫苗提供了实验基础.
목적 선취치병성구단라선체(간칭구체)중고도보수,동시시구체외막단백중함량최다적량개지단백LipL32화LipL21구건성융합기인DNA역묘pVAX1/LipL21-LipL32,관찰재BALB/c소서중중조DNA역묘유도면역응답반응적능력.방법채용련접인물PCR구건융합기인LipL21-LipL32,병장기삽입진핵표체재체구성중조DNA역묘pVAX1/LipL21-LipL32,지질체전염인배신세포(HEK293세포)후Western Blot험증중조DNA역묘재진핵세포중적표체,병장기기주BALB/c소서,용현미응집시험(MAT)검측소산생적특이성항체여문호구체적응집효개,용IL-10화TNF-β세포인자시제합검측체액면역화세포면역응답수평.결과 Western Blot분석현시중조DNA역묘pVAX1/LipL21-LipL32재HEK293세포중득도표체,소서동물실험결과현시중조DNA역묘능유효지유도궤체적체액면역화세포면역응답.결론성공구건구체융합기인LipL21-LipL32중조DNA역묘,소표체적융합단백능유도특이적면역응답반응,위진일보연구화발전신형적구체병역묘제공료실험기출.
Two highly conserved and with abundant quantity of lipoproteins in outer membrane of pathogenic species,but not in saprophytic species of leptospira ,LipL32 and LipL21,were selected to construct the fusion gene DNA vaccine pVAX1/LipL21-LipL32,and its ability to induce immune responses in BALB/c mice inoculated with this recombinant DNA vaccine was investigated in the present study.Expression of the fusion-protein LiPL21-LiPL32 was demonstrated in HEK293 cells following transfection with the fusion gene DNA vaccine and the immune responses induced after intramuscular inoculation with this DNA vaccine in BALB/c mice was then evaluated by microscopic agglutination test (MAT),meanwhile the ELISA assay was used to detect the cytokines induced.It was demonstrated that significant level of specific antibodies agglutinating antigens of Leptospira interrogans could be detected by MAT after DNA vaccine inoculation.The production of cytokines IL-10 and TNF-β in mice inoculated with DNA vaccine pVAX1/LipL21-LipL32 was significantly increased in comparison with that of the group inoculated with pVAX1 alone.These results indicate that the recombinant DNA vaccine pVAX1/LipL21-LipL32 may be of potential value to design and develop new generation of vaccines against leptospirosis.