ADV分离强毒株全基因突变重组质粒的构建、表达及其免疫原性的分析

ADV isolated virulent strain construction expression and immunogenicity analysis of whole gene mutation recombinant plasmids

关键词:  水貂阿留申病毒  真核表达载体  反应原性  免疫原性

Key words:  Aleutian disease of mink  Eukaryotic expression vector  Immunogenicity  reactionogenicity

摘要:

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质粒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质粒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,经肌肉注射重组质粒进行免疫, 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CD3+、CD4+和CD8+T淋巴细胞亚群。最后实验结果表明两组重组质粒具有良好的反应原性和免疫原性。

Abstract:

In order to develop the effective vaccines of Aleutian mink disease, in this experiment, the DNA sequence of the isolated virulent strain of Aleutian mink disease virus was used as a template, the 428-446 amino acid sequence of VP2 gene was removed by overlap extension PCR technique and connected with pcDNA3.1 ( ) carrier, constructed the of full gene mutation recombinant plasmid pcDNA3.1-ADV-428 At the same time, based on the removal of 428-446 amino acid sequence of truncated 487-501 amino acid sequence, to construct the full gene mutation recombinant plasmid pcDNA3.1-ADV-428-487. The recombinant plasmid was transfected into L929 cells and the expression was detected by indirect immunofluorescence (IFA) assay. To select healthy mice immunize with the recombinant plasmid by intramuscular injection, detect antibody by indirect ELISA method and detect the CD3 , CD4 and CD8 T lymphocyte subsets by flow cytometry in spleen cells from immunized mice at forty-second days.