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

为研制水貂阿留申病核酸疫苗,应用重叠延伸PCR技术去除ADV VP2基因中编码428~446位氨基酸的核苷酸序列,与pc DNA3.1(~+)载体连接,构建全基因突变重组质粒pc DNA3.1-ADV-428,在此基础上,截去编码487~501位氨基酸的核苷酸序列,构建全基因突变重组质粒pc DNA3.1-ADV-428-487。将构建的重组质粒经肌肉注射免疫小鼠,应用间接ELISA法检测接种后14、28、42、56 d抗ADV抗体水平;流式细胞术检测接种后第42天小鼠脾细胞CD3~+、CD4~+和CD8~+T淋巴细胞亚群。结果显示,小鼠接种质粒后CD3~+、CD4~+和CD8~+T淋巴细胞亚群数量均明显增加,第42天抗ADV抗体水平达峰值。本试验通过对ADV全基因突变重组质粒的免疫原性进行分析,为水貂阿留申病核酸疫苗的研制提供了参考。

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

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.