中国兽医科学2024，Vol.54Issue(3)：393-402,10.DOI:10.16656/j.issn.1673-4696.2024.0055
细粒棘球绦虫MDH基因的克隆表达结构功能预测及蛋白定位
Cloning,expression,structure and function prediction and protein localization of MDH gene of Echinococcus granulosus
摘要
Abstract
In order to explore the biological function of malate dehydrogenase(MDH)gene in the growth and development of Echinococcus granulosus and preliminarily evaluate the potential of MDH as a vaccine candidate antigen,the EgMDH gene sequence was obtained from the NCBI GenBank database,and the specific primers were designed.The full-length sequence of the MDH gene was amplified by PCR using the cDNA of the protoscoleces of E.granulosus as a template.The physicochemical properties and structural characteristics of MDH protein were analyzed by bioinformatics software.The recombinant plasmid pET28a-MDH was constructed and transformed into E.coli BL21(DE3)competent cells.The expression of the protein was detected by SDS-PAGE,then the protein was purified and refolded,and the antigenicity of the protein was analyzed by Western-blot.The localization of the target protein in the protoscoleces was detected by indirect immunofluorescence assay.The relative transcription level of EgMDHgene mRNA in protoscoleces and adults was analyzed by real-time fluorescence quantitative PCR.The results showed that the EgMDH gene was 999 bp in length,encoding 332 amino acids.The protein molecular formula was C1639H2599N4450475S16,the relative molecular weight was 36 651.32,and the theoretical isoelectric point was 8.11.The encoded protein had no signal peptide and transmembrane region,containing 40 phosphory-lation sites,3 N-linked glycosylation sites,and 9 dominant B cell epitopes.The secondary structure wascomposedof α-helix(46.69％),randomcoil(31.93％),extendedchainstructure(16.87％)andβ-turn(4.52％).The cloned gene was 975 bp in length with a predicted molecular weight of 35.7 ku.Western-blotting showed that the recombinant protein could be recognized by rabbit polyclonal antibody against His tag and mouse polyclonal antibody against soluble whole protein of protoscoleces.Indirect immunofluores-cence assay showed that EgMDH protein was localized in the capsule wall of protoscoleces.Real-time fluorescence quantitative PCR showed that EgMDH gene was expressed in both adults and protoscoleces,and the transcription level of protoscoleces was significantly higher than that of adults(P＜0.05).The results of this study preliminarily indicated that EgMDH has the potential to be a vaccine candidate antigen,in order to provide candidate antigens for the subsequent development of E.granulosus vac-cine;and also provided a basis for the further study of the MDHgene of E.granulosus.关键词
细粒棘球绦虫/苹果酸脱氢酶/生物信息学/原核表达/间接免疫荧光Key words
Echinococcus granulosus/malate dehydrogenase/bioinformatics/prokaryotic expres-sion/indirect immunofluorescence分类
农业科技引用本文复制引用
普娜,赵文卿,张玉霞,陈旭珂,张艳艳,孙艳,薄新文,王正荣..细粒棘球绦虫MDH基因的克隆表达结构功能预测及蛋白定位[J].中国兽医科学,2024,54(3):393-402,10.基金项目
国家自然科学基金项目(32360887,31860701) （32360887,31860701）
新疆生产建设兵团国际科技合作项目(2021BC008) （2021BC008）
省部共建绵羊遗传改良与健康养殖国家重点实验室重大专项(2021ZD02) （2021ZD02）
新疆生产建设兵团农业科技创新工程专项(NCG202213) （NCG202213）
