梅毒螺旋体外膜蛋白核酸疫苗的筛选构建及其免疫效果的比较研究

发布时间：2018-05-02 01:24

本文选题：梅毒螺旋体 + 外膜蛋白　；参考：《南华大学》2005年硕士论文

【摘要】：目的:筛选比较梅毒螺旋体(Treponema pallidum,Tp)各外膜蛋白的抗原性和同源性,分别构建真核表达重组体pcDNA3.1(+)-Gpd、pcDNA3.1(+)-Tp92和双基因融合表达重组体pcDNA3.1(+)-Gpd-Tp92,检测其所诱导产生的体液免疫和细胞免疫应答水平,比较单基因核酸疫苗和双基因融合核酸疫苗的免疫效果,为研制高效、新型的Tp核酸疫苗提供实验依据。方法:用PCGENE软件分析Tp外膜蛋白Gpd和Tp92基因序列,设计相应特异性引物,PCR分别扩增上述两基因1059bp和2103bp的目的基因片段,将其插入pUCm-T载体,通过蓝白斑初筛,酶切分析、PCR及测序鉴定筛选阳性重组体;将Gpd和Tp92基因亚克隆至真核表达载体构建pcDNA3.1(+)-Gpd、pcDNA3.1(+)-Tp92和pcDNA3.1(+)-Gpd-Tp92重组体,并分别转染HeLa细胞,用免疫细胞化学及western blot鉴定其在真核细胞的表达;分别在0、2、4、8w将核酸疫苗pcDNA3.1(+)-Gpd、pcDNA3.1(+)-Tp92、pcDNA3.1(+)-Gpd-Tp92及对照空质粒pcDNA3.1(+)分组注射于新西兰兔后腿股四头肌,每次剂量为100/μg,末次免疫后2w,无菌分离兔脾细胞,经特异性重组蛋白抗原刺激后,ELISA双抗体夹心法测定培养上清中IFN-γ水平,ELISA间接法测定新西兰兔血清中抗Gpd和抗Tp92特异性抗体水平;MTT法、淋巴细胞转化试验测定脾淋巴细胞特异性增殖反应。结果: 成功构建了pcDNA3.1(+)-Gpd和pcDNA3.1(+)-Tp92单基因真核表达载体
[Abstract]:Objective: to screen and compare the antigenicity and homology of the outer membrane proteins of Treponema pallidum TpP of Treponema pallidum. The eukaryotic expression recombinant pcDNA3.1 was constructed, and the recombinant pcDNA3.1was constructed respectively. The humoral and cellular immune responses induced by the recombinant plasmid pcDNA3.1and the double gene fusion recombinant pcDNA3.1were detected, and the immune effects of single-gene nucleic acid vaccine and double-gene fusion nucleic acid vaccine were compared. To provide experimental basis for the development of high-efficiency and novel TP nucleic acid vaccine. Methods: the Gpd and Tp92 gene sequences of TP outer membrane protein were analyzed by PCGENE software. Specific primers were designed to amplify the target gene fragments of 1059bp and 2103bp, respectively. The fragments were inserted into pUCm-T vector and screened by blue and white spot. Gpd and Tp92 genes were subcloned into eukaryotic expression vector to construct pcDNA3.1 (Tp92 and pcDNA3.1) and transfected into HeLa cells respectively, and their expression in eukaryotic cells was identified by immunocytochemistry and western blot. The nucleic acid vaccine pcDNA3.1 was injected into the quadriceps femoris hind leg of New Zealand rabbits at a dose of 100 / 渭 g at each dose of 100 / 渭 g at 8 weeks after the last immunization, and the splenocytes were isolated from rabbits by sterility 2 weeks after the last immunization, and the control plasmid pcDNA3.1was injected into the hind leg quadriceps of New Zealand rabbits, respectively, and the control plasmid pcDNA3.1was injected into the hind leg quadriceps of New Zealand rabbits at a dose of 100 / 渭 g each time, and the spleen cells were isolated from rabbits two weeks after the last immunization. The level of IFN- 纬 in culture supernatant was determined by Elisa double antibody sandwich method after stimulation with specific recombinant protein antigen. Indirect Elisa was used to detect the level of anti Gpd and anti Tp92 specific antibody in New Zealand rabbit serum. Lymphocyte transformation test was used to determine the specific proliferation of splenic lymphocytes. Results: The eukaryotic expression vectors of pcDNA3.1 (Gpd) and pcDNA3.1 (Tp92) were successfully constructed.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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