腺病毒41型感染致儿童腹泻一例临床表现及病毒基因组学特征分析  

Heterologous expression and purification of a novel oxidoreductase,Fnr,from Clostridium difficile

作　　者：王媛媛[1,2] 裴广倩 赵飞扬 范航 张湘莉兰 何鮦鮦 王云飞 米志强 安小平 黄勇 陈名武 童贻刚 WANG Yuan-yuan;PEI Guang-qian;ZHAO Fei-yang;FAN Hang;ZHANG Xiang-lilan;HE Tong-tong;WANG Yun-fei;MI Zhi-qiang;AN Xiao-ping;HUANG Yong;CHEN Ming-wu;TONG Yi-gang(Anhui Medical University,Hefei 230000,China;State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Microbiology and Epidemiology;The First Affiliated Hospital of USTC Anhui Provincial Hospital)

机构地区：[1]安徽医科大学,安徽合肥230000 [2]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室 [3]中国科学技术大学附属第一医院,安徽省立医院

出　　处：《中国病原生物学杂志》2018年第11期1236-1242,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81572045)

摘　　要：目的针对消化道疑似感染病例,采用高通量测序技术筛查病原体,并测定其全基因组序列,阐明其基因组及进化特征。方法通过对原因不明腹泻患儿的粪便进行病原体高通量基因测序,然后拼接其全基因组序列,采用最大似然法(选择bootstrap为1000)绘制主要抗原蛋白六邻体(hexon)、五邻体(penton)和纤维突蛋白(fiber)的进化树,分析进化特征;运用Recombination Detection Program version 4(RDP4)软件进行序列重组分析;使用软件CLC Genomics Workbench 9.0分析突变位点。结果患儿临床表现主要为发热、呕吐、腹泻伴抽搐,通过对腹泻患儿的粪便样品高通量测序,使用本实验室高通量测序数据分析程序检出41型腺病毒(HAdV-41)。进一步测序和拼接获得该病毒全基因组序列,其基因组全长34 138bp。序列比对显示该病毒核苷酸序列与已报道序列的最高同源性为99%(Human adenovirus 41isolate NIVD103),属于腺病毒F亚属。对该病毒六邻体、五邻体和纤维突蛋白进行进化分析,其六邻体与HAdV-41isolate NIVD103,HAdV-41isolate SH/2015/D16亲缘关系较近,五邻体与HAdV-41isolate NIVD103,HAdV-41isolate SH/2015/D16,HAdV41isolate SH/2015/D240,HAdV-41isolate SH/2015/D381亲缘关系较近,纤维突蛋白与五邻体相似;对全基因组序列进行重组分析,未发现明显的重组迹象,但突变分析显示引起此例腹泻的病毒株基因组有多处突变。结论从一例腹泻患儿粪便中检出的病原体为新的41型腺病毒,该病毒基因组与以往发现的41型腺病毒有较大差异,其流行态势、致病性及免疫原性值得关注。Objectives To ascertain the properties and function of the novel oxidoreductase Fnr fromClostridium difficile. Methods The fnr gene of Clostridium difficile was amplified using PCR.The PCR product was digested with the restriction endonucleases NheI and XhoI and inserted into pET28 b(+),which was similarly digested with restriction endonucleases,to construct the recombinant expression vector pET28 b(+)-fnr.The recombinant expression vector was verified as correct with sequencing and then transformed into E.coli C41(DE3)competent cells.Expression of the target protein was induced with IPTG.Finally,the recombinant Fnr protein was purified with affinity chromatography using an Ni-NTA column and its purity and enzyme activity were determined. Results The recombinant expression vector pET28 b(+)-fnr was successfully constructed and transformed into E.coli C41(DE3)to efficiently express recombinant Fnr.The recombinant protein was purified using affinity chromatography and then analyzed with SDS-PAGE,which indicated that the obtained protein was highly pure and consisted of two subunits with sizes as 51 kDa and 33 kDa.The purified protein displayed characteristic absorption at 390 nm and 430 nm and had specific activity at 30℃of 0.55 U/mg in the presence of NAD+according to a TTC reduction assay.In the absence of NAD+,only 30% was found.These enzymes exhibit the same properties,indicating that they perform the same biochemical function. Conclusion Recombinant FnrfromClostridium difficile was successfully expressed and purified.The protein displays the same oxidoreductase activity as displayed by other obligate anaerobic bacteria.This finding has laid the foundation for study of the physiological function of that oxidoreductase.

关 键 词：高通量测序 腺病毒41型 六邻体 五邻体 纤维突 

分 类 号：R373[医药卫生—病原生物学]