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

作　　者：普娜 赵文卿 张玉霞 陈旭珂 张艳艳[2] 孙艳[2] 薄新文[1,2] 王正荣[2] PU Na;ZHAO Wenqing;ZHANG Yuxia;CHEN Xuke;ZHANG Yanyan;SUN Yan;BO Xinwen;WANG Zhengrong(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding/lnstitute of Animal Husbandry and Veterinary Medicine,Xinjiang Academy of Agricultural Reclamation Sciences,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所,新疆石河子832000

出　　处：《中国兽医科学》2024年第3期393-402,共10页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32360887,31860701);新疆生产建设兵团国际科技合作项目(2021BC008);新疆生产建设兵团农业科技创新工程专项(NCG202213) 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);。

摘　　要：为了探究苹果酸脱氢酶(malate dehydrogenase,MDH)基因在细粒棘球绦虫(Echinococcus granulosus)生长发育中的生物学功能并初步评价MDH作为疫苗候选抗原的潜力,从NCBI GenBank数据库中获得Eg MDH基因序列,设计特异性引物,以细粒棘球绦虫原头蚴的cDNA为模板,用PCR扩增MDH基因全长序列;采用生物信息学软件对MDH蛋白的理化性质、结构特点等进行分析;构建重组质粒pET28a-MDH并转化大肠杆菌BL21(DE3)感受态细胞,利用SDS-PAGE检测蛋白的表达,随后纯化、复性蛋白,并通过Western-blot对蛋白进行抗原性分析;利用间接免疫荧光试验检测原头蚴中目的蛋白的定位;应用实时荧光定量PCR分析Eg MDH基因m RNA在原头蚴及成虫中的相对转录水平。生物信息学分析结果显示,Eg MDH基因长999 bp,编码332个氨基酸,蛋白分子式为C1639H2599N445O475S16,相对分子质量为36 651.32,理论等电点为8.11,且编码蛋白没有信号肽和跨膜区,含有40个磷酸化位点,3个N连接型糖基化位点,9个优势B细胞抗原表位。二级结构由α-螺旋(46.69%)、无规则卷曲(31.93%)、延伸链结构(16.87%)和β-转角(4.52%)构成。克隆的基因长975 bp,预测分子质量为35.7 ku;Western-blotting显示,重组蛋白可以被抗His标签的家兔多克隆抗体和原头蚴可溶性全蛋白小鼠多克隆抗体识别;间接免疫荧光试验显示,Eg MDH蛋白定位在原头蚴的囊壁;实时荧光定量PCR显示,Eg MDH基因在成虫及原头蚴中均有表达,且原头蚴阶段的转录水平显著高于成虫(P<0.05)。本研究结果初步表明Eg MDH具有作为疫苗候选抗原的潜力,以期为后续细粒棘球绦虫疫苗研发提供候选抗原;同时也为后续细粒棘球绦虫MDH基因的深入研究奠定了基础。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 Eg MDH gene sequence was obtained from the NCBI Gen Bank database,and the specific primers were designed. The full-length sequence of the MDH gene was amplified by PCR using the c DNA 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 p ET28a-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 Eg MDH gene m RNA in protoscoleces and adults was analyzed by real-time fluorescence quantitative PCR. The results showed that the Eg MDH gene was 999 bp in length,encoding 332 amino acids. The protein molecular formula was C1639H2599N445O475S16,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 phosphorylation sites,3 N-linked glycosylation sites,and 9 dominant B cell epitopes. The secondary structure wascomposedofα-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 immunofluorescence assay showed that Eg MDH protein was localized in the capsule wall of protoscoleces.Real-time fluorescence quantitative PCR sh

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 

分 类 号：S852.734[农业科学—基础兽医学]