利用展示尿素酶B表位的大肠杆菌菌蜕递送幽门螺杆菌核酸疫苗

探讨和分析了重组大肠杆菌菌蜕递送系统能否进一步提高幽门螺杆菌核酸疫苗的免疫应答水平.首先构建了展示尿素酶B表位的重组大肠杆菌菌蜕,然后以此菌蜕包裹幽门螺杆菌核酸疫苗pcDNAKAT,经DNA凝胶电泳、荧光显微镜观察和FACS分析,表明该质粒DNA可以非特异地结合到大肠杆菌菌蜕内膜上.该核酸疫苗经重组菌蜕包裹后免疫小鼠,其血清抗过氧化氢酶抗体效价由单独免疫时的1∶(307±39)提高到1∶(520±54),二者差异极显著(P=0.005 8),结果表明利用菌蜕递送系统能较好地提高核酸疫苗的免疫应答水平.     

制备禽致病性大肠杆菌菌蜕的三种方法比较

菌蜕(Bacterial ghosts)是一种只含有细菌内、外膜结构的细菌空壳,可作为新型疫苗和传递载体。本研究通过3种方式制备禽致病性大肠杆菌(Avian pathogenicity Escherichia coli,APEC)分离株DE17的菌蜕,评价不同的菌蜕制备方法。结果表明,利用噬菌体Phi X174的裂解基因E构建的溶菌质粒pBV220-E制备DE17菌蜕,对DE17菌株裂解率可达99.9%,扫描电镜观测结果表明,在DE17两端或中部形成可见的跨膜孔道,呈现典型的菌蜕结构。利用合成的细胞穿透肽MAP(KLALKLALKALKAALKLA)作用于DE17制备菌蜕,结果表明,10μmol/L的MAP可实现对OD_(600)=0.1的DE17完全灭活,扫描电镜虽未看到明显的跨膜孔道,但细菌的膜结构呈现沟壑状,而构建的可表达MAP的溶菌质粒pBV220-MAP并不能实现对DE17的裂解作用。本研究通过比较分析不同APEC菌蜕的制备方式,为进一步研究菌蜕疫苗和提高菌蜕疫苗的生物安全性提供参考。     

λpL/pR-cI857温控系统的改造及其对大肠杆菌菌蜕制备的影响

菌蜕是革兰氏阴性菌在噬菌体Phix174的溶菌基因E的作用下形成的细菌空壳,通常由λpL/pR-cI857温控系统通过调控E基因的表达制备。通过重叠PCR技术,将λpL/pR-cI857温控系统中λpR启动子的第2个操纵基因(OR2)的第9位碱基由T突变为C(T→C)。溶菌试验结果表明,突变后的λpL/pR-cI857系统在37℃时仍能稳定抑制基因E的表达,对大肠杆菌的溶菌率为99.9%。通过对λpR启动子的改造,扩大了λpL/pR-cI857温控系统的温度调控范围,为菌蜕疫苗的研制提供了参考。     

重组大肠杆菌菌蜕与幽门螺杆菌蛋白质疫苗协同免疫BALB/c小鼠

目的:考察重组菌蜕和蛋白质疫苗混合后对BAB/c小鼠机体免疫反应的影响。方法:首先构建展示尿素酶B抗原表位的大肠杆菌菌蜕,同时通过融合PCR构建分子内佐剂蛋白质疫苗rLTBKAT,然后分别利用该菌蜕和蛋白质疫苗免疫BAB/c小鼠。结果:当rLTBKAT和重组菌蜕联合口服免疫BAB/c小鼠时,其抗尿素酶B抗体的效价由单独免疫时的1∶(239±23)提高到1∶(681±76),二者差异极显著(P=0.009);而其抗过氧化氢酶抗体的效价则由单独免疫时的1∶(2800±275)下降至1∶(1800±400),二者差异不显著(P=0.08)。抗体分型试验表明,单独和联合免疫时,其抗尿素酶B抗体类型均以IgG1为主,而抗过氧化氢酶抗体类型则由单独免疫时的IgG1为主转变为联合免疫时的IgG2a为主。联合免疫时,小鼠抗尿素酶B和抗过氧化氢酶IgA抗体水平均高于单独免疫时。结论:重组菌蜕和蛋白质疫苗联合免疫,可提高机体对某些抗原的免疫水平或改变其反应类型。     

细菌菌蜕作为新颖药物递送体系的研究进展

细菌菌蜕是革兰氏阴性菌被噬菌体PhiX174的裂解基因E裂解后形成的完整细菌空壳.由于它具有完整的细菌表面抗原结构,所以它能直接作为疫苗使用.利用基因工程手段,可以非常便利地将外源抗原蛋白插入菌蜕的内膜、外膜或周质等多个部位,构建重组菌蜕多价疫苗.目前,菌蜕作为新颖的药物递送体系也开始受到关注.利用菌蜕可以递送DNA和蛋白质疫苗以及其他药物,能较好地产生免疫反应和治疗作用.     

菌蜕系统作为新型渔用疫苗体系的研究进展

菌蜕系统作为一种新型疫苗体系,是通过对噬菌体PhiX174裂解基因E的精确表达调控建立的.细菌菌蜕兼顾了组合抗原免疫原性、佐剂效应、靶向性载体等作用,特别适合于黏膜免疫及口服免疫;由于缺乏内含物而更加安全;生产过程简单,适宜大规模生产.这些特性决定了细菌菌蜕是一种具有良好应用前景的候选疫苗及递送系统.近年来随着水产养殖业的发展,鱼类细菌性疾病大规模爆发并造成严重损失.鉴于化学药物和常规疫苗的种种缺陷,新型渔用疫苗的研究日渐受到人们重视.在阐述细菌菌蜕系统形成和调控机制的基础上,着重介绍菌蜕疫苗在几种鱼类细菌性病害防治中的研究进展,并对菌蜕系统作为新型渔用疫苗体系的可行性和优越性进行讨论,对其应用前景进行展望.相信随着研究的深入,渔用菌蜕疫苗将在水产养殖病害防治中发挥着越来越重要的作用.     

迟缓爱德华菌菌蜕疫苗对罗非鱼注射和口服免疫的效果分析

通过腹腔注射、口服两种免疫途径探讨迟缓爱德华菌菌蜕疫苗对罗非鱼Oreochromis niloticus的免疫保护效果。将制备的迟缓爱德华菌菌蜕疫苗（ETG）和福尔马林灭活疫苗（FKC）采用腹腔注射、口服两种免疫途径免疫罗非鱼,分别于免疫后14d、21d和28d采集罗非鱼血清、头肾、脾脏,测定血清中抗体IgM水平,血清中酸性磷酸酶（ACP酶）、超氧化物歧化酶（SOD酶）活性及罗非鱼头肾和脾脏中白介素（IL-1）、肿瘤坏死因子（TNF）、干扰素（IFN）、Caspase3等细胞因子的相对表达量,并通过攻毒试验得到菌蜕疫苗、福尔马林灭活疫苗两种疫苗的相对免疫保护率。免疫组罗非鱼的血清抗体水平均极显著高于（P<0.01）对照组,ETG注射组抗体效价极显著高于（P<0.01）FKC口服组。免疫28d,免疫组SOD、ACP酶活力显著高于（P<0.05）对照组（Group E、F）;在头肾中,免疫组（Group A、B）TNF、IL-1和IFN的相对表达量显著高于（P<0.05）对照组（Group E、F）。在免疫保护试验中,所有免疫组的免疫保护率均显著高于（P<0.05）对照组,注射、口服菌蜕疫苗的相对保护率分别为79%、77%,注射、口服灭活疫苗组的的相对保护率分别为62%、60%,但两种疫苗免疫保护率无显著差异。试验研制的菌蜕疫苗得到更高的免疫保护率,菌蜕疫苗在预防罗非鱼爱德华菌病中有良好的研究价值和应用前景。     

迟钝爱德华氏菌菌蜕系统的构建

菌蜕系统（Bacterial Ghost,BG）的形成是利用噬菌体PhiX174的裂解蛋白E在革兰阴性菌细胞膜形成一个跨膜孔道结构,使细菌胞内物质由孔道排出而引起死亡。这种基因灭活的过程不引起细菌表面结构的任何理化变性,因此生成的细菌空壳具有与活菌相同功能的膜抗原结构,可诱导机体的体液免疫和细胞免疫应答。检测和比较了在铁调控启动子PyncE和温度调控启动子PR/cI控制下的E基因对迟钝爱德华氏菌菌蜕系统（EBG）的生成效率。结果显示,2种启动子均能成功生成EBG,电镜下可观察到细菌两端有直径约为80～400 nm的孔洞。传统菌蜕系统所用的热启动子在诱导后3 h开始裂解,8 h后细菌停止死亡;而新型铁诱导启动子在诱导后2 h细菌即完全停止生长。本研究为将来开发菌蜕载体疫苗防治爱德华氏菌症奠定了基础。     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.     

Mice vaccinated with enteropathogenic Escherichia coli ghosts show significant protection against lethal challenges

Aim: To prepare enteropathogenic Escherichia coli (EPEC) E2348/69 ghosts and investigate whether immunization with EPEC bacterial ghosts can elicit protective immune responses. Methods and Results: A recombinant plasmid with double λPL/PR‐cI857 temperature‐sensitive regulatory cassettes was constructed. The lysis gene E and/or the staphylococcal nuclease A (SNA) gene were separately inserted downstream of the two regulatory cassettes to construct the lysis plasmids pBV220::E and pBV220::E::CI‐P‐SNA. An EPEC reference strain E2348/69 (serotype O127:H6) was transformed with the lysis plasmids to produce EPEC ghosts. Mice injected with bacterial ghosts EGE (EPEC ghosts produced using lysis protein E) or EGES (EPEC ghosts produced using a combination of lysis protein E and SNA) gained weight normally and showed no clinical signs of disease. Vaccination trials showed that mice immunized with EGE or EGES were significantly protected against subsequent challenge with the wild‐type virulent parent strain, EPEC E2348/69 (42/50 and 45/50 survival, respectively); in contrast, none of the 30 control mice survived. Conclusions: Immunization with EPEC ghosts can elicit protective immune responses in BALB/c mice. Significance and Impact of the Study: EPEC ghosts may represent a promising new approach for vaccination against EPEC infection.     

Plasmid DNA production combining antibiotic‐free selection,inducible high yield fermentation,and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage λR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non‐ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid–liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts. Biotechnol. Bioeng. 2009; 104: 505–515 © 2009 Wiley Periodicals, Inc.     

Suppression of the Temperature-Sensitive Mutation of the bamD Gene Required for the Assembly of Outer Membrane Proteins by Multicopy of the yiaD Gene in Escherichia coli

We isolated temperature-sensitive mutants of the Escherichia coli bamD gene, which is essential for the assembly of β-barrel outer membrane proteins. As their multicopy suppressor, we identified a novel yiaD gene encoding a putative lipoprotein, YiaD. Mutations of its OmpA domain, which is required for interaction with peptidoglycan, affected suppression, suggesting that interaction with peptidoglycan is important to YiaD function.     

质粒拯救法克隆细菌基因组大片段

[目的]细菌基因组大片段尤其是基因簇的克隆与操作,是细菌基因功能分析的一个难点.基因组测序工作的不断完成和序列信息的大量积累,为细菌基因组:DNA的操作提供了方便.本文报道了利用细菌的全基因组信息和质粒拯救法的原理建立的一种克隆细菌基因组大片段的方法.[方法]首先,根据基因组序列信息,在待克隆片段的一侧扩增一段DNA,并将其克隆到自杀载体上构建打靶质粒,然后,将打靶质粒整合到细菌的基因组中构建重组菌,提取重组菌的基因组DNA,酶切,自连,转化,将自杀质粒与待克隆的目的片段一起拯救出来.最后,根据需要将拯救的DNA片段亚克隆到新的载体中.[结果]我们利用该方法克隆了布鲁氏菌中长度为11kb的virB操纵子,并构建了互补质粒.将该质粒导入到virB的突变株中后使virB操纵子的转录活性得到了恢复,表明该策略切实可行.[结论]这种重组克隆策略给我们提供了一种新的对细菌基因组大片段进行操作的方法.     

Survival of nonspecific porin-deficient mutants of Escherichia coli in black sea water

AIMS: The aim of this study was to investigate the links between survival of Escherichia coli in sea water microcosms in the laboratory and the presence of porins in the outer membrane. The E. coli strains studied were a wild-type strain and a series of outer membrane protein (omp) mutants. METHODS AND RESULTS: Bacteria were suspended in natural or filtered-autoclaved sea water microcosms and numbers determined over an incubation period by plate count and by count of cells capable of respiration. CONCLUSIONS: The type of omp mutation has a significant impact in bacterial survival. The double OmpC-OmpF mutant and the OmpR mutant (which was incapable of synthesizing OmpC and OmpF) survived poorly compared with single omp mutants and the wild-type strain. This suggests that these proteins are important in determining the entry of E. coli into the survival mode. The EnvZ mutant, which lacks the protein by which the cell senses some changes in the environment, survived as well as the wild-type strain when compared by plate counts and by respiring cell count. The loss of the EnvZ protein has no effect on survival but it could prevent the organism sensing the changes in the environment through which entry into the survival state is triggered. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is another piece in the puzzle as to how bacteria survive stress conditions.     

具有真细菌基因启动子活性的盐生盐杆菌质粒DNA片段

利用大肠杆菌启动子探测质粒ｐＫＫ２３２－８为载体、用两组限制性内切酶ＢａｍＨＩ－ＳａｌＩ和ＨｉｎｄⅢ－ＳａｌＩ分别消化盐生盐杆菌Ｊ７（Ｈａｌｏｂａｃｔｅｒｉｕｍｈａｌｏｂｉｕｍ）的质粒ｐＨＨ２０５,在体外进行重组,转化Ｅ．ｃｏｌｉＨＢ１０１感受态细胞,在含氨苄青霉素和氯霉素的选择平板上筛选转化子,并从随机挑选的２０株转化子中,获得抗氯霉素水平达到１１０μｇ／ｍｌ的转化子Ｔ１和Ｔ２,所含重组质粒分别被命名为ｐＪＨ和ｐＪＢ。经限制性酶切分析及杂交分析表明,ｐＪＨ质粒上插入了一段来源于ｐＨＨ２０５质粒的ＤＮＡ片段,其大小为８００ｂｐ左右。通过重新转化实验进一步表明,该ＤＮＡ片段在大肠杆菌中具有启动子功能,从而证明,在古细菌（盐生盐杆菌）的质粒ＤＮＡ中存在具有真细菌（大肠杆菌）基因启动子活性的ＤＮＡ片段。     

A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA

We detected Ent plasmids in 300 strains of human enterotoxigenic Escherichia coli, but one strain, E. coli 240-3, had neither a small nor a large plasmid and encoded the heat-labile enterotoxin (LTh(240-3)) gene on its chromosome. DNA sequences showed that LTh(240-3) differed by 12 and 14 base pairs from LT (LTh) and LT (LTp) from human H10407 and porcine EWD299 strains, respectively. In deduced precursor toxins, LTh(240-3), LTh and LTp differed from LTh, LTp and LTh(240-3) at nine, eight and eleven positions, respectively. These data suggest that although LTh(240-3) encoded in the chromosome is antigenically similar to LTh, it cannot be grouped with LTh due to differences in its DNA and amino acids sequences.