ADV全基因优化核酸疫苗的构建及其免疫效果的初步研究

发布时间：2018-09-04 08:24

【摘要】：水貂阿留申病(Aleutian disease,AD)是由水貂阿留申病毒(Aleutian disease virus,ADV)引起的一种侵害水貂免疫系统的疾病,又称为浆细胞增多症。由于其特殊的致病机理,导致目前尚无有效防治水貂阿留申病的疫苗。为了更好的研制有效对抗水貂阿留申病的疫苗,本实验以实验室所分离的水貂阿留申病强毒株DNA序列为模板,运用重叠延伸PCR技术,去除VP2基因中编码428-446氨基酸的核苷酸序列,并与pcDNA3.1(+)载体连接,构建全基因突变重组核酸疫苗pcDNA3.1-ADV-428;同时在去除428-446氨基酸序列的基础上截去487-501氨基酸序列,构建全基因突变重组核酸疫苗pcDNA3.1-ADV-428-487。将构建的重组质粒转染至L929细胞中,通过间接免疫荧光(IFA)实验检测其表达效果。选取健康小鼠分组,经肌肉注射进行免疫,用间接ELISA法检测抗体;在第42天取免疫小鼠的脾细胞用流式细胞仪检测、CD4+、CD8+和CD3+T淋巴细胞亚群;用酶联免疫检测试剂盒,检测免疫后小鼠血清中IL-4、IL-10、IL-12和IFN-γ四种细胞因子的含量。最后实验结果表明所构建的重组核酸疫苗具有良好的反应原性和免疫原性。将以上所构建的核酸疫苗经肌肉注射接种到水貂体内进行免疫。免疫完成后对水貂进行攻毒,然后每隔两周对水貂进行指尖采血。应用流式细胞术检测全血中的CD8+淋巴细胞亚群;应用间接ELISA法检测血清中的抗体水平;应用血清蛋白电泳检测血清中γ球蛋白占总蛋白的百分比以及应用聚乙二醇沉淀比浊法检测水貂血清中抗原抗体复合物的含量。实验结果表明pcDNA3.1-ADV-428和pcDNA3.1-ADV-428-487组在水貂试验中γ球蛋白、CIC以及CD8+T淋巴细胞的量都要低于对照组,且pcDNA3.1-ADV-428-487的保护效果要优于pcDNA3.1-ADV-428,所构建的核酸疫苗展现了良好的疫苗潜质。
[Abstract]:Mink Aleutian disease (Aleutian disease,AD) is a disease of mink immune system caused by mink Aleutian virus (Aleutian disease virus,ADV), also known as plasmacytosis. Due to its special pathogenic mechanism, there is no effective vaccine against Aleutian disease in mink. In order to develop an effective vaccine against Aleutian disease in mink, the DNA sequence of Aleutian virulent strain isolated in the laboratory was used as template, and the nucleotide sequence encoding 428-446 amino acid in VP2 gene was removed by overlapping extension PCR technique. The recombinant nucleic acid vaccine pcDNA3.1-ADV-428; was constructed by ligating with pcDNA3.1 () vector. The 487-501 amino acid sequence was cut off on the basis of removing 428-446 amino acid sequence, and the recombinant nucleic acid vaccine pcDNA3.1-ADV-428-487. was constructed. The recombinant plasmid was transfected into L929 cells and its expression was detected by indirect immunofluorescence (IFA) assay. Healthy mice were divided into groups and immunized by intramuscular injection, antibody was detected by indirect ELISA method, spleen cells of immunized mice were detected by flow cytometry and CD3 T lymphocyte subsets were detected by flow cytometry on the 42nd day, Elisa kit was used. The levels of four cytokines, IL-4,IL-10,IL-12 and IFN- 纬, in serum of immunized mice were determined. The results show that the recombinant nucleic acid vaccine has good reactivity and immunogenicity. The nucleic acid vaccine was injected intramuscularly into mink for immunization. After immunization, the mink is poisoned, and the mink is collected at the fingertips every two weeks. CD8 lymphocyte subsets in whole blood were detected by flow cytometry, antibody levels in serum were detected by indirect ELISA method. Serum protein electrophoresis was used to detect the percentage of 纬 globulin to total protein and polyethylene glycol precipitation turbidimetry was used to detect the content of antigen-antibody complex in mink serum. The results showed that the levels of 纬 globulin CIC and CD8 T lymphocytes in pcDNA3.1-ADV-428 and pcDNA3.1-ADV-428-487 groups were lower than those in the control group, and the protective effect of pcDNA3.1-ADV-428-487 was better than that of nucleic acid vaccine constructed by pcDNA3.1-ADV-428,.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.92

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