副猪嗜血杆菌OMP5的克隆表达及其对豚鼠免疫保护作用

根据GenBank中登录的副猪嗜血杆菌外膜蛋白P5(outer membrane protein P5,OMP5)基因序列设计1对特异性引物,以江西分离株NC0807基因组DNA为模板,扩增出OMP5基因。将其克隆到pET-28a(+)中,构建重组表达质粒pET-28a-OMP5,质粒转化大肠杆菌BL21(DE3),通过SDS-PAGE和Western blotting分析重组蛋白的表达情况和反应原性。重组蛋白经镍柱亲和层析纯化后免疫豚鼠,测定其免疫原性和保护效率。结果表明,重组蛋白在大肠杆菌中获得了高效表达。表达的蛋白分子质量约为43 ku,能被副猪嗜血杆菌阳性血清识别。动物试验结果表明,重组蛋白免疫后能诱导产生高水平的OMP5特异性抗体,并可显著保护豚鼠抵抗副猪嗜血杆菌强毒菌株的攻击,提示OMP5是副猪嗜血杆菌的保护性抗原。     

Immunological Identification and Characterization of Extracellular Serine Protease-Like Protein Encoded in a Putative espP2 Gene of Haemophilus parasuis

Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESP-like protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 10(9) bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection.     

Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis

The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA.     

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer's disease

Haemophilus parasuis is the etiological agent of Gl?sser's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Gl?sser's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness.     

副猪嗜血杆菌血清型和外膜蛋白P5基因型多样性的研究

副猪嗜血杆菌(Haemophilus parasuis,Hps)的血清型及基因型具有复杂多样性。本研究用间接血凝试验对某一猪场分离的Hps进行血清分型鉴定,并对部分菌株的外膜蛋白P5基因进行克隆测序和进化发育分析。试验结果显示,在21头病死猪不同部位共分离了42株Hps,其中 Hps血清5型17株(40.7%)、血清14型12株(28.5%)、血清4型1株(2.3%)和未定型12株(28.5%)。本试验中有5头猪同时感染2种不同血清型的Hps。21株Hps临床分离株分属5个不同基因序列型(STA～STE),其中STA有12株(12/21)为优势基因型;相同血清型的Hps分离株具有不同的STs型,来自同一头猪的Hps血清型和STs型也不相同。以上结果表明,同一猪群感染的Hps血清型和基因型存在明显的多样性,同一头猪感染Hps的血清型和基因型也存在多样性。本研究为进一步揭示猪群感染Hps的复杂性,研究该病的感染与流行机制提供了有价值的参考。     

Interactions of Haemophilus parasuis and its LOS with porcine brain microvascular endothelial cells

Haemophilus parasuis is a swine pathogen that causes Gl?sser's disease, which is characterized by polyserositis and meningitis. The pathogenesis of the H. parasuis infection is poorly understood. To cause meningitis, H. parasuis has to cross the blood-brain barrier (BBB) to gain access to the central nervous system (CNS). We recently showed that H. parasuis adheres to and invades porcine brain microvascular endothelial cells (PBMEC). The aim of this study was to evaluate the role of H. parasuis lipooligosaccharide (LOS) in the adhesion to PBMEC and to determine if H. parasuis (and/or its LOS) is able to induce apoptosis and activation of PBMEC. Results showed that adhesion of H. parasuis to PBMEC was partially mediated by LOS. Moreover, H. parasuis induces caspase-3-mediated apoptosis of PBMEC in a time--and dose--dependent manner, but its LOS did not seem to be involved in such a process. Furthermore, H. parasuis and, to a lesser extent, its LOS, was able to induce the release of IL-8 and IL-6 by PBMEC. Field strains of H. parasuis serotypes 4 and 5 induced similar levels of these inflammatory mediators. Our data suggest that H. parasuis uses cellular adhesion, induction of apoptosis and up-regulation of inflammatory mediators as mechanisms to invade the CNS via the BBB, and that LOS would play a certain but limited role in such pathological process.     

副猪嗜血杆菌rfaD基因的克隆及原核表达

rfaD基因编码ADP-L-甘油-D-甘露庚糖-6-异构体酶,缺失该基因会导致LOS糖链缩短和疏水性增强,从而影响细菌的致病性。为进一步探索副猪嗜血杆菌(Haemophilus parasuis,Hps)ADP-L-甘油-D-甘露庚糖-6-异构体酶的功能,本研究对Hps SC096 株rfaD基因进行克隆及原核表达。根据GenBank上登录的NC_011852序列,设计引物扩增rfaD基因,获得927 bp目的片段,将其克隆至pMD19-T载体。经送样测序鉴定正确后,连接到pET-32a(+)上进行原核表达,并用IPTG诱导,将诱导产物进行SDS-PAGE和Western blotting分析。SDS-PAGE结果显示,H.parasuis rfaD基因能在E.coli BL21(DE3)中表达,重组蛋白分子质量约为50 ku,与预期分子质量大小一致。Western blotting分析结果表明,该蛋白质能与H.parasuis血清4型阳性高免血清产生特异性结合反应,具较好的反应原性。     

副猪嗜血杆菌ompP2基因的克隆、表达及间接ELISA抗体检测方法的建立

用PCR方法扩增副猪嗜血杆菌的外膜蛋白ompP2基因,序列测定结果表明扩增片段全长1 188bp。将扩增片段插入表达载体pET-32a,转化BL21进行诱导表达,SDS-PAGE电泳结果证实重组融合蛋白约为60 000,West-ern-blot结果表明重组表达蛋白具有免疫反应性。以纯化的重组蛋白ompP2作为抗原,建立副猪嗜血杆菌的抗体间接ELISA检测方法。通过试验确定最佳反应条件为抗原包被质量浓度4.75mg/L,4℃包被过夜,待检血清的稀释浓度为1∶40,阳性判断标准为D630大于0.383。特异性和重复性试验表明,建立的间接ELISA方法具有良好的特异性和敏感性,可用于副猪嗜血杆菌的检测。     

Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor

Bacteria have evolved a set of highly specialized proteins to capture iron in iron-depleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Gl?sser disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under iron-restricted conditions, however, it is one of the targets of the humoral immune response.     

一株鸭源H6N6亚型禽流感病毒的全基因组序列分析及对小鼠的感染性研究

为了解H6N6亚型禽流感病毒(AIV)的生物学特性,本研究对2015年在广东活禽交易市场分离的一株鸭源AIV DK/GD/S1182/2015(H6N6)进行了全基因组测序、遗传演化分析和对BALB/c小鼠的感染性试验。序列分析显示,该病毒的HA蛋白裂解位点处仅有一个碱性氨基酸,符合低致病性AIV的分子特征;HA蛋白的222V和228S,可以增强病毒对α-2,6唾液酸受体的结合能力。NA蛋白颈部有11个氨基酸的缺失,这将会影响NA的神经氨酸酶活性;该病毒可能是2010年广东H6N6猪流感病毒与2014年广西AIV重组产生。小鼠感染性试验表明,该分离株不需要预先适应就能够在小鼠的肺脏内高效复制,提示该分离株具有感染哺乳动物的潜在风险。本研究对H6亚型AIV监测和相关生物学特性研究具有一定的指导作用。     

中国东南部地区副猪嗜血杆菌分离株ERIC-PCR指纹图谱分析

采用肠杆菌基因间重复一致序列PCR方法,在对15种副猪嗜血杆菌血清型参考株鉴定获得15种不同ERIC-PCR指纹的基础上,对分离自中国东南部发生Glasser's病的不同猪场的111株副猪嗜血杆茵进行了指纹鉴定.结果显示:111株分离株显示出23种指纹图谱,前3种最流行的指纹图谱为ERIC-PCR X X(20/111),X X ⅢⅠ(9/111)和Ⅳ(8/111).且在111株分离株中,来自不同地区的分离株分别表现出不同种类的指纹图谱.该试验表明,ERIC-PCR方法可适用于对某一地区的副猪嗜血杆菌进行分子流行病学的研究和基因型的鉴定;试验结果还揭示了副猪嗜血杆茵在中国东南部地区已广泛存在并具有多样的基因型.     

副猪嗜血杆菌广东分离株外膜蛋白P5基因的克隆及蛋白结构预测

根据GenBank上发表的副猪嗜血杆菌(HPS)外膜蛋白基因的核苷酸序列设计并合成1对特异性引物,从广东省HPS分离株外膜蛋白P5基因中扩增出与预期设计的1116bp大小相符的片段,将扩增产物连接到pMD18-T载体上,进行序列测定和分析。结果表明,克隆出HPS广东分离株的目的基因,核苷酸长为1116bp,共编码371个氨基酸,与已发表的HPS(SH0165)外膜蛋白基因核苷酸序列同源性100%,氨基酸同源性100%。生物学预测分析结果显示,HPS外膜蛋白是一种混合型结构蛋白,含有α-螺旋、β-折叠、β-转角和无规则卷曲,其β-转角和无规则卷曲区域可能形成抗原表位;其N端含有1个信号肽,最佳切割位点在21～22个氨基酸;有15个抗原决定簇;无跨膜区;同源建模分析,未见相似三维结构。     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model

The recently completed genome sequence of Haemophilus parasuis strain SH0165 allowed us to screen putative OMPs for the development of recombinant vaccines. The objective of this study was to evaluate the immunogenicity and protective efficacy of three OMPs of H. parasuis. Three putative OMPs (SmpA, YgiW and FOG) were cloned, expressed and purified by Ni affinity chromatography using nitriloacetic acid resin. Mice were immunized either individually (individual protein, IP) or synergistically (synergistic protein, SP) with the recombinant proteins. A significant increase in IgG titer was detected in all protein-immunized mice. Isotyping studies revealed that the antibodies produced were predominantly IgG2a-type, indicating a predominant Th1 response. A significant increase was observed in IL-2, IL-4 and IFN-γ levels in the culture supernatants of splenocytes isolated from immunized mice. Furthermore, mice were challenged intraperitoneally with 6×10(9)CFU (5×LD(50)) of highly virulent homologous serovar 5 strain (SH0165) or 7.0×10(9) CFU (5×LD(50)) of the heterologous serovar 4 strain (MD0322) at fourteen days after the last immunization. All of the recombinant proteins enhanced survival and reduced histopathological lesions. Our results indicated that the three OMPs showed protection both individually and synergistically against infection with the highly virulent H. parasuis in mice.     

乳胶凝集试验快速检测副猪嗜血杆菌

利用原核表达并经过纯化的副猪嗜血杆菌TbpB N端蛋白及人工合成的特异性多肽制备单克隆抗体,经碳化二亚胺(EDC)将纯化后的单克隆抗体IgG与羧化乳胶共价偶联,并对偶联乳胶的IgG含量及偶联时间等条件进行合理选择,建立了快速检测副猪嗜血杆菌的乳胶凝集试验方法.利用LAT方法检测148株副猪嗜血杆菌疑似菌株,参考16 S rRNA-PCR方法的结果,符合率为83.1%,且致敏乳胶不与临床常见菌株发生凝集反应.结果表明,此检测方法具有操作简便、快速、特异性高的特点,便于在基层推广使用,为副猪嗜血杆菌的诊断和有效防治提供了参考和依据.     

Recombinant expression of porcine spermadhesin AWN and its phospholipid interaction: Indication for a novel lipid binding property

AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three‐step purification protocol based on different chromatographies. The test for AWN–phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation‐induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm–spermadhesin interaction and might provide new approaches for artificial reproduction techniques.     

Haemophilus parasuis exhibits IgA protease activity but lacks homologs of the IgA protease genes of Haemophilus influenzae

Haemophilus parasuis, the bacterium responsible for Gl?sser's disease, is a pathogen of significant concern in modern high-health swine production systems but there is little information regarding the identity or function of its virulence factors. Several important human mucosal pathogens, including the closely related bacterium Haemophilus influenzae, utilize IgA proteases to aid in defeating the host immune response and facilitate disease but it is unknown whether H. parasuis synthesizes any product with IgA protease activity. To investigate potential virulence mechanisms of H. parasuis, we evaluated five strains for their ability to digest purified IgA. Western blotting demonstrated cleavage of swine IgA, but not human IgA1, following incubation with culture supernatants from three strains, two of which are known to cause invasive disease. No genes with homology to the H. influenzae IgA protease genes iga and igaB could be identified in any H. parasuis strain using either PCR or Southern blotting. These results demonstrate that a novel IgA protease produced by some strains of H. parasuis cleaves the swine IgA heavy chain at a site not found in human IgA1.     

副猪嗜血杆菌CDT亚基原核表达及其抗原性鉴定

为原核表达副猪嗜血杆菌(H.parasuis)CDT毒素并检测其在体外培养时的分泌表达情况,本实验将切除信号肽的CDT毒素的3个亚基基因分别克隆于pET-28a载体中在大肠杆菌进行表达,并将重组蛋白经亲和层析纯化后,免疫兔制备抗血清,用于鉴定H.parasuis体外培养时CDT分泌表达情况。结果表明CDT毒素3个亚基均在Rosetta菌株中得到表达,其中cdtB约为28.2 ku,符合预期大小,而cdtA和cdtC亚基比预期的23.5 ku和17.4 ku略大。表达的3个重组蛋白可以被自然感染猪血清识别,表明其具有良好的反应原性,同时也表明H.parasuis在猪体内定殖或感染过程中分泌CDT。制备的抗血清可以与体外培养H.parasuis的分泌蛋白中的CDT亚基反应,表明重组蛋白具有良好的免疫原性,同时也表明H.parasuis体外培养时也分泌CDT。     

Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene

On the basis of a species-specific PCR assay, a RFLP analysis for typing of Haemophilus parasuis strains was developed and evaluated. Amplification was based on the gene tbpA, encoding a transferrin-binding protein. RFLP analysis of the 1.9-kb tbpA-amplicon using TaqI, AvaI and RsaI endonucleases produced 12 different patterns for the reference strains of the 15 known H. parasuis serovars, and showed a high heterogeneity (33 RFLP groups) for 101 H. parasuis clinical isolates tested. The sensitivity, typeability (100% versus 65% for immunodiffusion), high degree of discrimination (0.93 versus 0.84 for immunodiffusion), simplicity and low cost per test make this PCR-RFLP assay a useful method for typing H. parasuis and, therefore, for studying the epidemiology of outbreaks of Gl?sser's disease.