表达猪附红细胞体ENO基因重组腺病毒的构建及免疫原性分析

doi:10.16431/j.cnki.1671-7236.2019.07.025

Abstract

Abstract:

The aim of the experiment was to construct a recombinant adenovirus expressing Mycoplasma suis ENO gene and analyze its immune effect.Recombinant cloning plasmid pMD-19T-ENO and adenovirus shuttle vector AdV4-GFP were double digested to construct recombinant adenovirus shuttle plasmid AdV4-M/ENO;The recombinant adenovirus shuttle plasmid AdV4-M/ENO,which was linearized by PacⅠ,was transfected into 293 cells to obtain recombinant adenovirus Ad4-M/ENO.Expression of Mycoplasma suis ENO gene in 293 cells was identified by PCR and indirect immunofluorescence assay (IFTA),293 cells were cultured and the titer of recombinant adenovirus was determined.30 BALB/c mice were divided into three groups:Recombinant adenovirus Ad4-M/ENO group,AdV4-GFP empty vector control group and PBS control group,respectively,vaccinated separately.ELISA was used to detect IgG,IgG₁,IgG_2a antibody levels and IFN-γ and IL-4 cytokine levels in serum,and the levels of CD4⁺ and CD8⁺ in the spleen of mice were detected 2 weeks after the third immunization.The results showed that the recombinant adenoviral shuttle plasmid AdV4-M/ENO gene fragment size was 1 182 bp;The recombinant adenovirus Ad4-M/ENO was successfully packaged and expressed in 293 cells with a titer of 1×10⁹ PFU/mL.The serum levels of IgG,IgG₁,IgG_2a,IFN-γ,IL-4 and lymphocyte subsets CD4⁺ and CD8⁺ in BALB/c mice immunized with recombinant adenovirus Ad4-M/ENO were significantly or extremely significantly higher than AdV4-GFP empty vector control group and PBS control group (P<0.05;P<0.01).The results showed that the recombinant adenovirus expressing Mycoplasma suis ENO gene was successfully constructed in this experiment,and the recombinant adenovirus could induce specific humoral and cellular immune responses in mice.

Key words: Mycoplasma suis; ENO gene; recombinant adenovirus; immune effect

CLC Number:

S858.28

YAN Kexin, WU Shengjun, ZHAO Yun, WANG Miao, XU Yingtian, XUE Shujiang. Construction and Immunogenicity Analysis of Recombinant Adenovirus Expressing Mycoplasma suis ENO Gene[J]. , 2019, 46(7): 2088-2095.

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Construction and Immunogenicity Analysis of Recombinant Adenovirus Expressing Mycoplasma suis ENO Gene

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