马链球菌兽疫亚种PepO蛋白原核表达及其免疫保护试验

以马链球菌兽疫亚种(Streptococcus equi subsp.zooepidemicus,SEZ)ATCC35246株基因组为模板,根据NCBI数据库已公布的pepO基因序列设计引物进行扩增,利用同源重组技术连接至pCold-SUMO质粒进行原核表达及纯化,并以纯化的蛋白制备兔抗SEZ PepO多克隆抗体.将兔...     

马链球菌兽疫亚种类M蛋白亚单位疫苗小鼠免疫试验

为获得控制猪链球菌病安全高效的疫苗，本试验进行了马链球菌兽疫亚种类M蛋白亚单位疫苗的研制。应用热酸法提取马链球菌兽疫亚种ATCC35246株类M蛋白，并分别采用羟基磷灰石（HAT）层析和冷酒精沉淀法进行纯化，结果得到较纯的类M蛋白。用热酸提取的粗制类M蛋白、2种纯化的类M蛋白及全菌苗免疫小鼠，结果保护率分别为92％（11／12）、100％（12／12）、100％（12／12）和83％（10／12），表明类M蛋白亚单位疫苗有进一步开发的必要。     

马链球菌兽疫亚种的保护性抗原SzP蛋白的鉴定与评价

马链球菌兽疫亚种是引起猪链球菌病的主要病原之一。该菌的致病机理尚不清楚,且缺乏合适的疫苗,使猪链球菌病很难得到有效的控制。本实验通过构建SzP重组表达载体,纯化重组蛋白,对其的免疫效力进行了评价。结果表明该蛋白可以诱导高滴度的血清IgG抗体,并且可提供一定的免疫保护效力。进一步研究表明该蛋白是一个重要的体内诱导抗原,可诱导高水平的Thl和Th2型免疫应答。揭示了SzP蛋白在致病过程中起到重要作用,为新型疫苗的研制及致病机制的研究奠定了基础。     

马链球菌兽疫亚种ATCC35246酮基转移酶的原核表达及其免疫保护试验

根据已发表的马链球菌兽疫亚种MGCS10565酮基转移酶(transketolase)的基因序列,设计并合成引物。以ATCC35246株基因组DNA为模板,通过PCR技术,扩增出目的基因并定向克隆至表达载体pET-28a(+)中,然后将重组质粒转化入大肠杆菌BL21(DE3)中,分析并纯化表达产物。选用ICR小鼠作为实验动物模型,以纯化的重组融合蛋白通过皮下注射途径免疫小鼠,并用间接ELISA法监测小鼠血清中的抗体效价。结果表明重组蛋白免疫小鼠后能产生有效的免疫应答,血清中抗体水平有明显的升高。加强免疫2周后,以5LD50的ATCC35246强毒株攻击免疫组及对照组,结果免疫组小鼠的保护率可达37.5%。表明原核表达产物免疫ICR小鼠,可使其对同源菌株攻击产生一定的保护作用,在亚单位疫苗研制中具有潜在的应用价值。     

马链球菌兽疫亚种SeseC-00619蛋白的功能研究

为了阐明马链球菌兽疫亚种新蛋白SeseC-00619(细胞表面蛋白,cell surface protein,CSP)的功能,本试验采用real-time qPCR分析CSP的体内诱导表达特征,同时在大肠埃希菌中表达与纯化马链球菌兽疫亚种表面相关蛋白CSP,研究其免疫反应性及免疫保护功能,并试图解释其分子机制。通过流式细胞术与黏附抑制试验探索CSP对小鼠肺上皮细胞的黏附作用。结果表明,CSP是一个重要的体内诱导抗原,并且能在S.zooepidemicus细菌表面表达,属于细菌表面蛋白。重组蛋白CSP能够黏附LA-4细胞表面,而这种黏附作用可以竞争性抑制S.zooepidemicus黏附宿主细胞。本结果为进一步研究CSP基因的功能及其在致病机制中的作用奠定了基础。     

马链球菌马亚种3种抗原蛋白对小鼠免疫的免疫原性分析

旨在对比研究马链球菌马亚种(Streptococcus equisubsp.equi,S.equi)3种不同抗原蛋白——分选酶A(sortase A,SrtA)、M蛋白(SeM)及IgG结合蛋白(EAG)的免疫原性及免疫保护效果,本研究以表达并纯化的Sr-tA、SeM及EAG 3种蛋白单独及联合免疫小鼠,免疫后检测了血...     

马链球菌兽疫亚种毒力因子研究进展

马链球菌兽疫亚种(Streptococcus equi ssp.Zooepidemicus,SEZ)属于兰氏分类C群,旧称兽疫链球菌,普遍存在于动物的黏膜,尤其是马属动物的皮肤、上呼吸道黏膜、扁桃体以及生殖道等处。可致多种家畜的炎症及败血病。毒力因子是指构成细菌毒力的物质。马链球菌兽疫亚种具有类M蛋     

马链球菌马亚种8组分融合蛋白的表达及小鼠免疫效果的评价

马腺疫是由马链球菌马亚种(S. equi)引起的急性接触性马属动物传染病,严重影响幼驹的生长发育。我国尚无预防该病的有效疫苗,也缺乏S. equi动物感染和免疫保护模型。为在原核系统中表达马链球菌马亚种(S. equi)的多组分融合蛋白,并评价其对小鼠的免疫保护效果,本研究以S. equi HLJ2018株DNA为模板,分别经PCR扩增其cne、eag、SclC、SclI、SclF、eq5、eq8和ideE基因,并分别克隆至pGEX-6p-1载体中,构建表达这8个蛋白的重组质粒并分别经PCR和测序鉴定。通过在各基因之间引入蛋白接头Gly-Gly-Gly编码基因序列并利用同源重组技术依次串联该8个基因,得到融合基因se8,利用其构建重组质粒pET-28a-SE8 (His标签)及pGEX-6p-1-SE8(GST标签),均经PCR和测序鉴定后,将这两个表达SE8及分别表达该8个蛋白的重组质粒分别转化大肠杆菌BL21(DE3),经IPTG诱导表达后采用亲和柱层析法纯化,采用SDS-PAGE检测各蛋白的表达及纯化效果;采用western blot检测各蛋白的反应原性。SDS-PAGE结果显示...     

马链球菌兽疫亚种hasB基因功能的研究

为了阐明马链球菌兽疫亚种(Streptococcus equi ssp.zooepidemicus,SEZ)荚膜的致病机理,使该菌引起的猪、马、牛等多种动物的链球菌病得到有效控制,试验采用Real-time PCR技术分析has B基因的体内表达特性,通过构建基因缺失突变菌株、调理吞噬试验等对has B基因的致病性进行系统评价。结果表明:相对于体外培养的细菌,SEZ感染小鼠脾脏分离细菌的mRNA表达均显著下调;基因缺失突变菌株Δhas B与亲本菌株的生长状况基本一致,但是致病力显著降低;基因缺失突变菌株Δhas B影响补体和巨噬细胞介导的免疫反应,使其抵抗宿主巨噬细胞的吞噬能力显著降低。     

马链球菌兽疫亚种SzP的功能研究

为了阐明马链球菌兽疫亚种免疫原性蛋白Sz P的功能,试验采用Real-time PCR分析Sz P的体内诱导表达特征,同时在大肠埃希菌中表达与纯化马链球菌兽疫亚种表面相关蛋白Sz P,研究其免疫保护功能,并试图解释其保护机制,通过流式细胞术与黏附抑制试验探索Sz P对小鼠肺上皮细胞的黏附作用。结果表明:Sz P是一个重要的体内诱导抗原,并且能在S.zooepidemicus表面表达,属于细菌表面蛋白。重组蛋白Sz P能够黏附于LA-4细胞表面,而这种黏附作用可以竞争性抑制S.zooepidemicus黏附宿主细胞。说明Sz P在S.zooepidemicus黏附侵入宿主过程中起到重要作用。     

Identification of a novel protective antigen, 3-oxoacyl-[acyl-carrier-protein] synthase II of Streptococcus equi ssp. zooepidemicus which confers protective effects

Streptococcus equi ssp. zooepidemicus (SEZ) is an important swine pathogen and responsible for a wide variety of infections in many animal species. FabF was a novel protein identified in the previous study. However, its protective efficacy remained to be evaluated. In this study, recombinant fabF of SEZ was expressed and showed a strong immunoreactivity with mini-pig convalescent sera. Study in mice revealed that the recombinant protein induced a marked antibody response and protected 80% of mice against SEZ infection. The hyperimmune sera against fabF could efficiently kill the bacteria in the phagocytosis test. In addition, it was also found that anti- fabF antibodies can significantly inhibit the formation of SEZ biofilm. These study suggest that fabF may represent immunogens of interest for vaccine development against SEZ infection.     

Identification of Streptococcus equi ssp. zooepidemicus surface associated proteins by enzymatic shaving

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is responsible for a wide variety of infections in many species. Attempts to control the infection caused by this agent are hampered by a lack of effective vaccines and useful diagnostic kits. Surface proteins of bacterial species are usually involved in interaction with host and hopefully act as biomarkers for serodiagnosis and subunit vaccine components. In this study, the surface proteins of SEZ C55138 strain were systematically identified by surface shaving with trypsin and a total of 20 surface associated proteins were found. Further analysis of five selected novel proteins (SzM, FBP, SAP, CSP and 5'-Nu) revealed that they all expressed in vivo and their recombinant derived proteins could be reactive with convalescent sera. These identified immunogenic surface proteins have potential as SEZ vaccine candidates and diagnostic markers.     

A case of zoonosis associated with Streptococcus equi ssp. zooepidemicus

A sexagenarian couple operating a dairy farm, where there were also many horses, suffered a severe respiratory infection associated with the bacterium Streptococcus equi ssp. zooepidemicus. The investigation revealed that the source of infection was due to contacts with a horse having an infected wound from which we isolated this zoonotic agent.     

Characteristics of an R antigen common to Streptococcus equi and zooepidemicus

An R antigen of the group C streptococcus S. equi that cross reacts with a similar antigen of S. zooepidemicus has been identified and characterized. It is acid, heat and trypsin resistant, but pepsin sensitive and has an isoelectric point of 4.8. The amino acids in highest concentration are glutamic, aspartic, alanine, leucine, and valine. Bacterial components released in a French Press contain large amounts of R antigen, which is present also in culture supernatants and acid extracts. It has a molecular weight of about 82,000. Trypsin extraction of cells yields molecules of predominantly 56,000 and 25,000 molecular weight that appeared by immunoblotting to be similar to those obtained by trypsinization of purified R protein from preparations derived from the French Press. Although horses naturally infected with S. equi or S. zooepidemicus develop cross reacting R antibodies, the role of the R antigen in pathogenesis is unknown. Purified R protein does not stimulate bactericidal antibody in horses nor is it protective for mice. Its occurrence in antigen preparations in assays for S. equi antibody could be a source of interpretive errors because of the presence in many sera of antibody to the immunologically similar R antigen of S. zooepidemicus, a normal nasopharyngeal commensal of Equidae.     

Isolation and further characterization of phase variants of Streptococcus equi subsp. zooepidemicus

In the present study the soft agar technique was used to isolate phase variants of S. equi subsp. zooepidemicus-cultures isolated from infections of horses. The phase variants were characterized by a compact or diffuse colony morphology in this media. The variants could be cultivated separately and further characterized genotypically by RAPD analysis and by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, indicating the identity of both strains of each pair. The diffuse colony variants grew uniformly turbid after cultivation in fluid media, did not haemagglutinate rabbit erythrocytes, and displayed a reduced surface hydrophobicity in hexadecane and phenyl-sepharose adherence tests. The compact colony variants generally grew as sediment with clear supernatant in fluid media, haemagglutinated rabbit erythrocytes and showed an enhanced surface hydrophobicity in both hydrophobicity tests. The presented soft agar technique allowed a demonstration of phase variation of S. equi subsp. zooepidemicus and a subsequent isolation of the variants. This might be an important prerequisite to understanding the pathogenic importance of phase variation among isolates of this bacterial species.     

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.