鸭C4BPα的克隆、表达及其与鸭疫里默氏菌的互作

为研究鸭C4结合蛋白(C4b-binding protein,C4BP)与鸭疫里默氏菌(Riemerella anatipestifer,RA)的相互作用,对鸭C4BPα进行克隆、原核表达,免疫小鼠制备多克隆抗体,并利用间接免疫荧光试验及斑点杂交试验验证C4BP与RA的相互作用。结果显示,鸭C4BPα核苷酸序列全长为1230bp,与鸡C4BPα的相似性最高(82.1%);系统进化树分析发现,鸭C4BPα与鸡C4BPα处于同一系统进化树分支上,两者遗传进化关系最近;C4BPα在大肠杆菌Escherichia coli BL21 (DE3)中能高效表达,重组蛋白以胞内可溶性形式存在;多克隆抗体效价超过1∶10000,并且可以与重组蛋白发生特异性反应;间接免疫荧光试验和斑点杂交试验结果显示RA与鸭C4BP可以发生相互作用。研究结果为进一步揭示RA的致病机制奠定了基础。     

鸭疫里默氏杆菌铁离子代谢机制研究进展

铁离子是大多数细菌生存所必需的一种营养物质,但过多的铁离子会通过芬顿反应产生的活性氧对细菌造成损伤。因此,细菌通过摄取、调控、螯合、外排等机制维持体内铁离子的稳态。鸭疫里默氏杆菌(Riemerella anatipestifer)是一种最新被归类于威克斯菌科里氏杆菌属的革兰氏阴性菌。该菌主要感染禽类,参与该菌的铁离子代谢基因具有特别之处。本文对鸭疫里默氏杆菌铁离子代谢机制研究进展进行了系统总结和阐述,包括该菌的TonB系统、TonB依赖性受体、Fur蛋白及Dps蛋白等在铁离子转运、调控、螯合中的功能,以及以上蛋白在鸭疫里默氏杆菌致病中的作用,以期更全面地理解鸭疫里默氏杆菌铁代谢机制,并为进一步深入研究该菌铁离子代谢提供理论依据和参考。     

鸭疫里默氏杆菌RecA作为一种内参蛋白的评价

由于缺少适合的内参蛋白,所以用Western blot的方法来研究鸭疫里默氏杆菌蛋白表达的调节尚未见报道。以鸭疫里默氏杆菌基因组为模板,PCR扩增含有组氨酸标签His-tag的rec A基因和截段的rec AP基因,并分别将其克隆于原核表达载体p ET32a。将重组质粒转化到表达菌Rosetta中,经1mmol/L的IPTG诱导进行蛋白质表达。用组氨酸标签亲和试剂镍-琼脂糖进行重组蛋白质的纯化。用纯化的重组蛋白对4周龄的昆明鼠进行免疫,制备多克隆抗体。Western blot实验表明,截段表达的Rec AP的多克隆抗体血清比全长Rec A蛋白的多克隆抗体血清与鸭疫里默氏杆菌总蛋白反应更特异;在铁离子限制性环境和血红素丰富环境中培养的鸭疫里默氏杆菌的Rec A蛋白表达量一致。因此,鸭疫里默氏杆菌的Rec A蛋白可以在铁离子和血红素代谢等研究中作为Western blot的内参蛋白。     

鸭疫里氏杆菌hemH基因功能初步鉴定及其缺失株的转录组学分析

血红素是绝大多数细菌生长繁殖所必需的一种营养物质,其参与了细菌多种重要的生理过程。细菌可以通过自身合成和从外界摄取2种方式获得血红素。然而过多的血红素对细菌是有毒性的,细菌则会利用外排、螯合和降解等多种方式减轻血红素毒性。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染禽类的革兰氏阴性菌,前期研究表明该菌可通过转运的方式从外界环境摄取血红素。然而,是否该菌也可以自身合成血红素未知。基因组分析发现鸭疫里氏杆菌ATCC11845菌株中的基因RA0C＿RS08070编码铁螯合酶(ferrochelatase) HemH,是参与将铁插入卟啉中心,形成血红素的关键酶。hem H缺失后会导致铁离子和卟啉的积累,对细菌造成毒性。【目的】为鉴定Hem H在合成血红素中的功能及查找参与鸭疫里氏杆菌铁离子和卟啉解毒相关基因。【方法】本研究构建了RAATCC11845Δhem H缺失株,并检测亲本株和hem H缺失株在GCB以及GCB添加血红蛋白液体培养基中的生长情况;随后对亲本株和hem H缺失株进行转录组测序并进行比较分析。【结果】RAATCC11845Δhem H缺失...     

鸭疫里默氏杆菌Mtan对底物SAH的催化活性

【目的】分析鸭疫里默氏杆菌(Riemerella anatipestifer,RA)不同血清型pfs基因的序列差异,并开展其编码蛋白S-腺苷高半胱氨酸核苷酶(Mtan,又称Pfs)的催化活性研究。【方法】PCR扩增9株不同血清型RA的pfs基因,分析其核苷酸序列的同源性;构建该基因的重组表达载体p Cold-RA-pfs,表达、纯化RA的重组蛋白Mtan(RA-Mtan);测定RA-Mtan对底物S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)的催化活性,运用哈维弧菌报告菌株BB170检测催化底物的自诱导物2(Autoinducer-2,AI-2)活性。【结果】对RA的pfs序列分析结果表明,不同血清型RA的核苷酸一致性在93.9%-100%之间;SDS-PAGE检测结果表明,RA-Mtan呈可溶性表达;酶活测定表明RA-Mtan和禽致病性大肠杆菌(Avain pathogenic Escherichia coli,APEC)的Lux S蛋白共同作用于底物时,可产生浓度为176.7μmol/L的同型半胱氨酸(Homocysteine,HCY);AI-2活性检测结果表明,产生的AI-2具有生物学活性。【结论】RA不同血清型的pfs高度保守,RA pfs基因的编码产物RA-Mtan在体外具有催化SAH的活性,RA-Mtan和禽致病性大肠杆菌的Lux S蛋白共同作用于底物SAH时,能产生有活性的AI-2,为进一步研究pfs对RA的调控作用提供参考。     

利用16SrDNA建立种特异性PCR快速检测鸭疫里默氏菌

鸭疫里默氏菌感染是危害养鸭业的主要疾病,用表型指标鉴定鸭疫里默氏菌存在不足,因此有必要建立检测该菌的种特异性PCR法。利用已登录的鸭疫里默氏菌、大肠杆菌、沙门氏菌、多杀性巴氏杆菌的16S rDNA基因序列,设计了一对鸭疫里默氏菌16S rDNA基因的特异性引物190f和843r,分别以基因组DNA和菌落提取液为模板,从1-19型鸭疫里默氏菌参考菌株和代表亚型、变异株和可能新型的国内分离株共26株细菌中均扩增出大小为654bp的特异性片段,而扩增鸭大肠杆菌、鸭沙门氏菌和禽多杀性巴氏杆菌等感染鸭的常见细菌的结果均呈阴性。分别将鸭疫里默氏菌基因组DNA和菌落提取液进行10倍梯度稀释,基因组DNA的最小检出量为50pg,菌落最小检出量为15CFU／mL。结果说明,该PCR法具有较好的特异性和敏感性,可用于快速鉴定鸭疫里默氏菌。     

Identification of a virulence-associated protein homolog gene and ISRa1 in a plasmid of Riemerella anatipestifer

Riemerella anatipestifer is the causative agent of polyserositis of ducks and geese. We have previously reported that a 3.9-kb plasmid, pCFC1, carries protein genes (vapD1 and vapD2) that are similar to virulence-associated genes of other bacteria. In the present study, we report the complete sequence of a second plasmid of 5.6 kb, pCFC2. pCFC2 has a 28% G-C content and three large open reading frames (ORFs). One of the ORFs (designated asVapD1) encodes a polypeptide that shares 53.9, 53.9, 48.3, 48.3 and 46.1% identity with virulence-associated proteins of Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae, Helicobacter pylori and Haemophilus influenzae, respectively. The second ORF encodes a putative DNA replication protein (RepA3) with 309 amino acids and a molecular mass of approximately 36 kDa. A novel insertion sequence (IS) element, designated ISRa1, was found on the plasmid pCFC2. ISRa1 was flanked by 15-bp imperfect inverted repeats (only one mismatched nucleotide). ISRa1 contained an ORF encoding a putative transposase of 292 amino acids. Southern blot analysis indicated that in R. anatipestifer strains examined, ISRa1 was present with 2-20 copies (at least). ISRa1 displayed a sequence approximately 35% homologous to the putative IS982 and RSBst-alpha from Lactococcus lactis ssp. cremoris SK11 and Bacillus stearothermophilus CU21. Three hybridization patterns of genomic DNA of eight R. anatipestifer strains with an ISRa1 probe indicated that ISRa1 might be a useful tool for epidemiological studies.     

鸭疫里默氏菌DnaK、OmpA和OmpA-DnaK蛋白的免疫原性

【背景】鸭疫里默氏菌(Riemerella anatipestifer,RA)可引起鸭等多种禽类败血症和浆膜炎,给禽养殖业造成严重经济损失。蛋白疫苗是预防RA感染的重要策略之一。目前,有关RA重组蛋白免疫原性报道较少,且其应用也受到单一蛋白抗原诱导的特异性免疫反应不足的限制。【目的】探究分子伴侣DnaK、外膜蛋白A (outer membrane protein A,OmpA)和OmpA-DnaK蛋白疫苗在鸭体内诱导的免疫应答,评估其免疫原性,为RA疫苗抗原研发提供依据。【方法】克隆DnaK和OmpA基因并分别与pET-32a(+)载体相连,利用限制性酶切位点Nco I和Bam H I将OmpA连接至DnaK基因上游,经原核表达和纯化制得重组蛋白DnaK、OmpA和OmpA-DnaK。3种重组蛋白分别皮下免疫雏鸭2次,检测其血清抗体滴度、淋巴细胞增殖和细胞因子(IL-2和IL-4)水平;以RA-GH5肌肉注射攻毒,检查其组织病理学变化及免疫保护率。【结果】成功表达了DnaK、OmpA和OmpA-DnaK重组蛋白,分子量分别约为90、60和130 kDa。3种蛋白疫苗均能诱导宿主产生体液...     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.