中国农业大学学报2024，Vol.29Issue(11)：102-111,10.DOI:10.11841/j.issn.1007-4333.2024.11.11
锡兰钩虫ASP4基因的表达与免疫保护效果分析
Expression of ASP4 gene in Ancylostoma ceylanicum and immunprotective effect analysis of recombinant protien
摘要
Abstract
To clone the ASP4 gene fromAncylostoma ceylanicum(Ace-ASP4)and evaluate the immune protective effect induced by its reconbinant protein in mice,the ASP4 gene sequence was firstly amplified,and the amino acid sequence of its enconded protein was subjected to bioinformatics analysis.The recombinat protein was then expressed in prokaryotic cells to immunize mice.The proliferatin abilitily of mouse spleen lymphocyte proliferation ability and serum IgG,IgG1 and IgG2a antibody levels were detected using CCK-8 and indirect ELISA methods,respectively.In addition,the levels of IFN-γ,IL-2,IL-4 and IL-10 in the serum of mice after the first immunization were also detected.Finally,after three immunizations,the L3 larvae were orally infected at 500 larvae per mouse 48 h later.The number of larvae in the lungs,gastrointestinal tract,and muscles were counted 48 h post-infection,and the reduction rate was calculated to evaluate its immunoprotective effect.The results showed that:1)The ORF of Ace-ASP4 gene was 750 bp long,encoding 249 amino acids.Bioinformatics analysis showed that Ace-ASP4 encoded a hydrophilic protein with a signal peptide sequence and no transmembrane structure,and its three-dimensional structure model had a good quality with a GMQE value of 0.9.2)The molecular weight of Ace-ASP4 protein was 27 ku,and it could be expressed both in the supernatant and precipitate,but mainly enriched in the formed of inclusion bodies in the precipitate and had good immunoreactivity.3)The Ace-ASP4 recombinant protein can induce significant increase of IgG,lgG1 and IgG2a antibodies in immunized mice and promote the proliferation of spleen lymphocytes in immunized mice(P＜0.001).Compared with the PBS control group,levels of IFN-γ,IL-2,IL-4 and IL-10 in mice immunized with Ace-ASP4 recombinant protein on the 35th day after the first immunization were significantly increased(P＜0.05).4)After challenge,compared with mice immunized with PBS,mice immunized with 50 µg/mL of recombinant protein achieved a 52％reduction rate in the lungs,a 46％reduction rate in the gastrointestinal tract,and a 61％reduction rate in the muscles.In conclusion,Ace-ASP4 protein was successfully expressed,and can induce strong immune response in immunized mice,which lays a foundation for further study of the biological function of ASP4 gene and provides reference data for the development of A.ceylanicum vaccine.关键词
锡兰钩虫/ASP4基因/原核表达/免疫保护效果分析Key words
Ancylostoma ceylanicum/ASP4 gene/prokaryotic expression/immune protection effect分类
农业科技引用本文复制引用
聂林冲,袁浩,杨子鹏,何厚劲,李文豪,杨留禄,金梦圆,袁子国..锡兰钩虫ASP4基因的表达与免疫保护效果分析[J].中国农业大学学报,2024,29(11):102-111,10.基金项目
广东省自然科学基金项目(2023A1515011795) （2023A1515011795）
国家自然科学基金项目(31972707) （31972707）
广东省林业科学院项目(2023CG171) （2023CG171）
