胸膜肺炎放线杆菌血清10型apxIC-/p36+弱毒株的构建与鉴定

摘要：【目的】构建血清10型胸膜肺炎放线杆菌弱毒菌株,为胸膜肺炎放线杆菌减毒活疫苗研究奠定基础。【方法】通过细菌接合转移和SacB负向筛选标记完成突变株的构建与筛选,用PCR、Western blot、重组位点序列对突变株进行鉴定分析。首先构建含肺炎支原体p36基因的pEICALDH重组转移质粒,并转化供体大肠杆菌( E. coliX7213),将转化的阳性克隆子与野生型APP血清10型亲本菌混合培养6 h;然后涂至含氯霉素抗性和烟酰胺腺嘌呤二核苷酸（NAD）的TSA培养基培养,挑取阳性克隆,接种至无抗性的含NAD的TSB液体培养基,培养6～8 h后涂至含10%的蔗糖及NAD的TSA培养基,培养24 h后挑取蔗糖抗性的克隆,即得到目的突变株。【结果】小鼠毒力试验结果表明突变株比亲本株的毒力显著降低;生长特性分析结果显示突变株与亲本株的增殖能力无显著差异;同时免疫试验结果表明突变株与安全剂量的亲本株均可诱导小鼠产生较好的免疫反应,证明apxIC基因缺失并不影响APP的免疫活性。【结论】成功构建了含猪肺炎支原体p36基因的胸膜肺炎放线杆菌血清10型突变株,所获得的突变株有望成为猪传染性胸膜肺炎弱毒疫苗株。     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA₇, was sequenced. The amino acid sequence of OmlA₇ showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA₁) and 5 (OmlA₅), respectively. The first 134 amino acids of OmlA₇ were identical to those of OmlA₅. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA₇ in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA₇ detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.     

产琥珀酸放线杆菌发酵生产琥珀酸的研究进展

近年来,因瘤胃微生物产琥珀酸放线杆菌Actinobacillus succinogenes具有高的琥珀酸产量,并能够利用多种碳源进行发酵等优点,在利用发酵法生产琥珀酸领域具有广泛的应用前景和商业化价值,因而其代谢途径和发酵工艺等基础研究成为国内外研发的热点。近年来,人们在产琥珀酸放线杆菌的代谢途径、琥珀酸发酵动力学模型、新型经济培养基以及高产菌株选育等方面的研究取得了很大进展,对研发琥珀酸发酵工艺、降低生产成本和节能减耗等具有重要的理论意义。     

一株琥珀酸产生菌的筛选及鉴定

从牛的瘤胃中筛选获得一株能发酵生产琥珀酸的兼性厌氧菌。对其进行生理生化特性鉴定及16S rRNA基因分析。该菌株短杆状,无鞭毛,革兰氏染色阴性,V-P反应阴性,能发酵多种糖类产酸;其16S rRNA基因与琥珀酸放线杆菌的同源性高达99．8%,认为属于琥珀酸放线杆菌(Actinobacillus succinogenes),并将其命名为琥珀酸放线杆菌(Actinobacillus succinogenes)SW0580,保藏号CGMCC 1593。初步发酵试验表明该菌能发酵60g/L葡萄糖产生25．8g/L的丁二酸。     

Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors, ApxIIA, a bacterial exotoxin, is reportedly expressed in many serotypes and is considered as a candidate for the development of a vaccine against the bacterial infection. Previously, we isolated a field strain of A. pleuropneumoniae serotype 2 in Korea and characterized its exotoxins to develop an oral vaccine. In this study, we initially confirmed the immunogenicity of ApxIIA expressed in Escherichia coli. We then developed transgenic tobacco expressing ApxIIA and tested its efficacy to induce a protective immune response against A. pleuropneumoniae infection after oral administration of the plant powder. We observed that protective immune responses were induced in mice after oral administration of the plant powder once a week for 4 weeks. Immunoassays revealed that the levels of antigen-specific immunoglobulin G against ApxIIA increased in mice that were fed a powder made from the transgenic plant, but not in mice fed a powder made from wild-type tobacco. Additionally, mice fed the transgenic plant powder were protected from an injection of a lethal dose of A. pleuropneumoniae. These results support that the transgenic plant may be a suitable candidate for an oral vaccine that could be used effectively against A. pleuropneumoniae infection.