【作者】 汪文玉；

【导师】 谭立志；

【作者基本信息】 南华大学 ， 病原生物学， 2006， 硕士

【摘要】 目的；构建钩端螺旋体外膜脂蛋白lipL21基因的真核表达载体pcDNA3.1(+)-lipL21，直接肌注免疫豚鼠，观察其在豚鼠体内所产生的体液免疫和细胞免疫应答水平，研究其对豚鼠的兔疫保护作用，为研制高效的钩端螺旋体新型疫苗提供实验依据。 方法：根据GenBank钩端螺旋体外膜脂蛋白lipL21基因的基因序列AY187271，设计相应特异性引物，PCR扩增目的基因片段(561bp)，纯化回收后将其插入pUCm-T载体，通过蓝白斑筛选，酶切分析、PCR鉴定筛选阳性重组体；将lipL21基因亚克隆至真核表达载体pcDNA3.1(+)，构建pcDNA3.1(+)-lipL21重组体，将其转染HeLa细胞，用免疫细胞化学及SDS-PAGE分析鉴定lipL21在HeLa细胞中得到表达后，于0w、2w、4w将核酸疫苗pcDNA3.1(+)-lipL21及对照空质粒pcDNA3.1(+)和对照PBS分别经豚鼠右后腿股四头肌肌肉注射免疫豚鼠，每次剂量为100μg／只，末次免疫后2w，以培养10d的56601株培养物从腹腔攻击各免疫组豚鼠(2mL／只)，连续观察15d，观察各免疫组豚鼠的发病情况，并取各免疫组豚鼠的肺、肝、肾做病理切片，HE染色观察比较各免疫组豚鼠的组织病理变化，分析重组体pcDNA3.1(+)-lipL21对豚鼠的免疫保护作用。并无菌分离豚鼠脾淋巴细胞，经非特异性抗原植物血凝素(PHA)刺激后，MTT法和淋巴细胞转化试验测定各免疫组豚鼠脾淋巴细胞的体外增殖情况，显微镜凝集试验(MAT法)测定各免疫组豚鼠血清中抗LipL21抗体水平，PCR法检测lipL21基因在免疫豚鼠肌细胞内是否存在。 结果：成功扩增出lipL21基因，并构建了真核表达重组质粒pcDNA3.1(+)-lipL21，重组质粒转染HeLa细胞48h后，免疫细胞化学结果显示在HeLa细胞胞浆内有棕黄色的阳性产物，而pcDNA3.1(+)转染的HeLa细胞胞浆内未观察到棕黄色的阳性产物，SDS-PAGE结果显示目的基因能在HeLa细胞表达Mr约21kDa的蛋白。MAT结果显示更多还原

【Abstract】 Objectives:(1) To construct Eukaryotic Expression vector pcDNA3.1 (+)-lipL21 containing the outmembrane lipoprotein lipL21 gene of Leptospira and to express target protein in HeLa cells.(2) To observe immune response induced by DNA vaccine on the out membrane lipoproteinLipL21 of Leptospira in guinea-pigs,(3) To observe the protective effects of DNA vaccine on the out membrane lipoprotein LipL21of Leptospira in guinea-pigs from the infection by pathogenic Leptospira.Methods:Primers were designed by primer primierS.O software according to the lipL21 gene sequence of Leptospira interrogans serovar Lai. Polymerase chain reaction (PCR) was used to amplify the lipL27-encoding gene and about 561 bp lipL21 gene fiagment was isolated. The fragment of lipL21 was directly ligated into pUCm-T .After identified by PCR and restriction enzyme analysis, the fragment of lipL21 gene was subcloned into eukaryotic expression vector pcDNA3.1 (+) with linking reactions to construct pcDNA3.1 (+)-lipL21 The recombinant plasmid was transfected into HeLa cells using liposome .After expression of the lipL21 gene in HeLa cells was identified by immunocytochemistry and SDS-PAGE ,2-weeks old guinea-pip were immunrzed with pcDNA3.1(+)-lipL21 or pcDNA3.1(+) or PBS intramuscularly at 2 week interval for three times. MAT was used for the quantitative detection of the specific antibody in the sera of immune guinea-pigs. The proliferation response of spleen cells was detected by MTT and lymphocyte transformation experiment The existence of lipL21 gene in muscle of guinea-pigs was detected by PCR.Results: The lipL21 gene fragment (561bp) was successfully amplified. The eukaryotic更多还原