新孢子虫硫氧还蛋白的生物信息学分析及其原核表达的鉴定  

Bioinformatics analysis and prokaryotic expression,purification and identification of Thioredoxin in Neospora caninum

作　　者：齐闻新 张旻[1] 李晨 周思含 徐啊慧 钱伟锋[1] 胡苏辉 王天奇[1] 位治国[1] 洪炀 闫文朝[1] QI Wen-xin;ZHANG Min;LI Chen;ZHOU Si-han;XU A-hui;QIAN Wei-feng;HU Su-hui;WANG Tian-qi;WEI Zhi-guo;HONG Yang;YAN Wen-chao(College of Animal and Technology,Henan University of Science and Technology,Luoyang 471023,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,NHC Key Laboratory of Parasite and Vector Biology,Shanghai 200241,China)

机构地区：[1]河南科技大学动物科技学院,河南洛阳471023 [2]中国疾病预防控制中心寄生虫病预防控制所国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,上海200241

出　　处：《中国预防兽医学报》2024年第7期691-699,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31702226、U2004151);河南科技大学博士科研启动基金(13480069)。

摘　　要：为研究新孢子虫硫氧还蛋白(Trx)的生物学特性,本研究利用PCR扩增犬新孢子虫Trx(NcTrx)基因的编码区序列,构建pET-32a(+)-NcTrx原核表达载体,经双酶切与测序鉴定正确后,利用系列生物信息学软件分析NcTrx基因及其编码氨基酸序列的生物信息学特征,并对其蛋白互作网络中排名前100的蛋白进行GO功能和KEGG信号通路的富集分析。重组质粒中NcTrx基因的测序结果显示,NcTrx基因片段为1 287 bp,编码428个氨基酸;该蛋白含1个信号肽,1个跨膜区,主要位于内质网上;NcTrx蛋白的二、三级结构主要由α-螺旋、无规则卷曲组成,有12个B细胞抗原表位;该蛋白属于硫氧还蛋白家族,存在典型的氧化还原基序“-CXXC-”,有38个磷酸化位点、10个O-糖基化位点;与NcTrx蛋白互作最强的为酪氨酸激酶样蛋白,二者通过氢键和静电作用实现相互对接;GO功能及KEGG信号通路的显著性富集分析结果显示,NcTrx与其互作的蛋白主要参与蛋白质转运、蛋白质二硫键异构酶活性等生物学过程,与内质网蛋白质加工、硫代谢等信号通路密切相关。将pET-32a(+)-NcTrx转化BL21(DE3)感受态细胞,并经IPTG诱导表达后采用镍亲和层析法纯化重组NcTrx蛋白(rNcTrx),经SDS-PAGE检测该蛋白的表达及纯化效果,采用Bradford法测定纯化蛋白的浓度。采用western blot鉴定rNcTrx的反应原性。SDS-PAGE结果显示,rNcTrx分子量约66 ku,主要以包涵体的形式表达,且纯化效果较好,浓度为0.8 mg/mL。Western blot检测结果显示,纯化的rNcTrx能够与鼠His MAb、新孢子虫感染的小鼠血清发生特异性反应,在66 ku处出现特异性条带。本研究首次经PCR扩增NcTrx基因,对NcTrx基因及其蛋白进行了生物信息学分析,并经原核系统表达及鉴定了NcTrx蛋白,为后续深入探究该蛋白的生物学功能提供参考依据。To investigate the biological characteristics of Neospora caninum thioredoxin (Trx),the N.caninum Trx (NcTrx) gene was amplified by PCR,and the p ET-32a(+)-NcTrx prokaryotic expression vector was constructed and characterized by doubleenzyme digestion and sequencing.The NcTrx gene and its deduced amino acid sequences were analyzed using bioinformatics software,and the resulting protein networks were subjected to GO function and KEGG signaling pathway enrichment analysis.The pET-32a(+)-NcTrx was transformed into BL21(DE3) host bacterium.The recombinant NcTrx protein was purified by nickel affinity chromatography after induction with IPTG,and the expression and purification were examined by SDS-PAGE.The concentration of purified protein was determined by Bradford methord.The reactivity of rNcTrx protein was identified by western blot.The results showed that NcTrx gene fragment was 1287bp,encoding 428 amino acids.The protein included a signal peptide and a transmembrane region,primarily located on the endoplasmic reticulum.Its secondary and tertiary structure were mainly composed of α-helices and random coil,containing 12 B-cell antigenic epitopes.The NcTrx protein belonged to the thioredoxin family and possessed the characteristic redox motif'-CXXC-'.It had 38 phosphorylation sites and 10 O-glycosylation sites.The NcTrx protein exhibited the highest interaction with TKL proteins through hydrogen bonding and electrostatic interactions.The significant enrichment analysis of GO function and KEGG signaling pathway showed that NcTrx and its interacting proteins were mainly involved in biological processes such as protein transport and protein disulfide bond isomerase activity,playing crucial roles in endoplasmic reticulum protein processing,sulfur metabolism,and other signaling pathways.SDS-PAGE results showed that rNcTrx was mainly expressed as inclusion body with a molecular weight of approximately 66ku,and was purified at a concentration of 0.8mg/mL.Western blot results demonstrated that the purified protein was abl

关 键 词：新孢子虫 硫氧还蛋白 生物信息学 原核表达 

分 类 号：S855.9[农业科学—临床兽医学]