生物法合成伤寒O-糖蛋白结合疫苗及其免疫原性评估

伤寒由伤寒沙门氏菌(Salmonella Typhi)引发,至今在发展中国家仍是备受关注的重要公共卫生问题。文章通过敲除伤寒菌脂多糖合成途径中O-抗原连接酶基因,转入含脑膜炎奈瑟球菌(Neisseria meningitidis)蛋白糖基化途径中糖基转移酶的表达载体,以及改构的重组铜绿假单胞菌(Pseudomonas Aeruginosa)外毒素A(rEPA_N29)的表达载体,使细胞内能够诱导合成以伤寒O特异性多糖(O-specific polysaccharides, OPS)为目标抗原、以rEPA_N29为载体蛋白的伤寒OPS-rEPA_N29糖蛋白复合物,并对纯化所得复合物进行了免疫原性评价。ELISA测定血清抗体滴度表明,rEPA_N29作为载体蛋白能有效增加糖链的免疫原性,糖蛋白比单独的多糖能诱导产生更好的免疫应答;3次免疫、间隔3周比间隔2周IgG滴度稍有提高;而免疫过量的糖蛋白,抗O-多糖的血清抗体效价并无提升。文章为生物法制备多糖-蛋白结合疫苗提供了新思路,理论上也适用于其他革兰氏阴性菌的疫苗研发。     

大肠杆菌O141 O-抗原基因簇的破译及进化分析

对大肠杆菌O141 O-抗原基因簇进行测序,序列全长15601bp,用生物信息学的方法进行序列分析,共发现12个基因：鼠李糖合成酶基因(rmlB,rmlD,rmlA,rmlC)、甘露糖合成酶基因(manB,manC),糖基转移酶基因(orf6,orf7,orf9,orf10)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy)。用PCR的方法筛选出了针对大肠杆菌O141的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O141的快速检测。通过对大肠杆菌O141的O-抗原基因簇及甘露糖和鼠李糖合成酶基因的进化分析发现：大肠杆菌O141 O-抗原基因簇是低GC含量的片段,仅O-抗原特异的基因才出现在O-抗原基因簇;并且这些基因可能介导了O-抗原基因簇间的重组及以O141 O-抗原基因簇的形成。     

大肠杆菌O23 O-抗原基因簇序列的破译及UDP-N-乙酰葡萄糖-C4异构酶的鉴定

采用鸟枪法破译大肠杆菌O23标准株的O-抗原基因簇序列,并用生物信息学的方法进行了基因注释和分析;采用基因缺失和互补的方法鉴定了O23的UDP-GlcNAc C4异构酶(Gne);用同源建模的方法构建了O23 Gne的高级结构并对其活性位点进行了分析;分析了不同血清型大肠杆菌O-抗原基因簇中gne基因的多样性;根据O23O-抗原基因簇中的特异基因筛选出了可用于大肠杆菌O23快速检测的特异DNA序列。     

国际沙门氏菌中心1984年修订的沙门氏菌属抗原表简介

本文就国际沙门氏菌中心1984年修订的沙门氏菌抗原表作了简单介绍。并对沙门氏菌属分类命名近况作了讨论。本抗原表采用了Le Minor分类命名系统,将沙门氏菌拟分为6个亚种。由于某些0群的取消及菌型的合并,本抗原表截止1983年底,共载有2107个菌型。     

大肠杆菌O54 O-抗原基因簇的破译及进化分析

破译了大肠杆菌O5 4O 抗原基因簇的序列 ,序列全长 1 4 0 6 2bp。用生物信息学方法分析序列并鉴定基因 ,共确定 1 0个基因 ,包括鼠李糖合成酶基因BDA和C(rmlBDA和rmlC) ,糖基转移酶基因 ,O 抗原转运酶基因 ,O 抗原聚合酶基因和合成磷酸丝氨酸侧链的基因及 1个不能确定功能的开放阅读框。对rmlC的 (G C) %含量 ,稀有密码子含量及进化分析都表明大肠杆菌O5 4O 抗原基因簇是在近期通过rmlC介导的重组形成 ,而且大肠杆菌O5 4和鲍氏志贺氏菌 9型的亲缘关系很近。对UTP 葡萄糖 1 磷酸 尿苷转移酶基因 (galF)和 6 磷酸葡萄糖脱氢酶基因(gnd)的进化分析揭示志贺氏菌属与大肠杆菌属在进化上属于同一个属。用PCR方法筛选出了针对大肠杆菌O5 4的特异基因 ,用于基因芯片或PCR方法对大肠杆菌O5 4的快速检测。     

禽流感抗原表位在沙门氏菌菌毛的表达

目的:经抗原表位预测和同源性比较,禽流感M1蛋白位于58-66序列的九肽是A型流感病毒中保守并具有很强免疫原性的T细胞表位,鉴于鼠伤寒沙门氏菌LT2的SEF17菌毛基因agfA作为疫苗载体的优势,在其上构建引起机体细胞免疫的沙门氏茵口服活体重组疫苗,以求在人类对抗禽流感过程中发挥作用。方法:利用两步重叠延伸PCR和基因置换,将外源表位插入LT2茵毛,并利用抗生素抗性,温度敏感质粒,及茵毛的刚果红吸附能力筛选菌毛上插有外源抗原表位的重组菌,并通过测序进一步验证外源基因的插入。结果:两步重叠延伸PCR产物AB,CD,AD长度与理论大小530bp,423bp,932bp一致。两次转化PCR鉴定,产物长度与理论大小932bp,634bp一致。刚果红吸附测定,菌毛上插入有外源肽的菌落因吸附刚果红能力减弱呈粉色,对筛选出的KmS型粉色茵落的PCR鉴定,产物长度与理论大小417bp一致,测序结果也显示agfA中外源表位基因的插入。结论:禽流感M1蛋白位于58-66序列的T细胞表位成功插入沙门氏茵SEF17菌毛基因agfA。     

减毒沙门氏菌作为DNA疫苗载体的研究进展

随着沙门氏菌基因组学的深入研究以及DNA重组技术的发展,使得对沙门氏菌进行精确的不可回复性的基因缺失减毒成为可能。减毒沙门氏菌可作为DNA疫苗载体,特异性地将其携带的质粒DNA靶向性的传递给巨噬细胞、树突细胞等抗原递呈细胞,从而有效激发相应的体液与细胞免疫应答。减毒沙门氏菌已作为疫苗载体在针对细菌、病毒、寄生虫等的DNA疫苗研究中得以广泛应用。     

伤寒沙门氏菌及其抗原检测方法的近年进展

伤寒病的诊断,来自病原学的证据最为可靠。检测伤寒特异性抗体,对诊断有重要的参考价值,但特异抗原刺激机体产生有关抗体,有一定的时间过程,而且受各种因素影响。在一些免疫功能紊乱或缺陷的患者中,检测抗体往往易产生假阳性或假阴性反应,而检测抗原则可     

The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses

Abstract The rpoS (katF) gene, which encodes a RNA polymerase σ factor ( σ ^s ), regulates the virulence of Salmonella typhimurium in mice. In the present study, we show that rpoS mutants can be frequently found among laboratory strains of Salmonella . In addition, a rpoS mutation was identified in the S. typhi live oral vaccine Ty21a. Introduction of a wild-type rpoS gene in Ty21a allowed the bacteria to survive better under starvation conditions and increased their resistance to other stresses. These results contribute to a better understanding of the genetic background of the live typhoid oral vaccine Ty21a and suggest that the rpoS mutation may contribute to the safety of this strain in humans.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by ¹³C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.

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where L-Rhap3Rlac2Ac is 2-O-acetyl-3-O-[(R-1-carboxyethyl]-L-rhamnose

     

Bacterial antigen polysaccharides. 43. Structure for the O-specific polysaccharide of <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O46

Acidic O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. The following structure of the hexasaccharide repeating unit of the O-specific polysaccharide was established using methylation analysis along with ¹H and ¹³C NMR spectroscopy, including 2D ¹H, ¹H-COSY, TOCSY, ROESY, ¹H, ¹³C-HSQC, and HMQC-TOCSY experiments:

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The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.     

Structure and gene cluster of the O-antigen of Escherichia coli O19ab

The O-polysaccharide (O-antigen) of Escherichia coli O19ab was studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit was established:→2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-d-Glcp-(1→3)-α-d-GlcpNAc6Ac-(1→where the degree of O-acetylation of GlcNAc is ∼33%. The O-antigen gene cluster of E. coli O19ab was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the E. coli O19ab-antigen structure.     

Towards more virulent and antibiotic-resistant Salmonella?

Salmonella are well-known pathogens. Virulence determinants can be present on the chromosome, usually encoded on pathogenicity islands, or on plasmids and bacteriophages. Antibiotic resistance determinants usually are encoded on plasmids, but can also be present on the multidrug resistance region of Salmonella Genomic Island 1 (SGI1). Virulence plasmids show a remarkable diversity in the combination of virulence factors they encode, which appears to adapt them to specific hosts and the ability to cause gastroenteritidis or systemic disease. The appearance of plasmids with two replicons may help to extend the host range of these plasmids and thereby increase the virulence of previously non- or low pathogenic serovars. Antibiotic resistance among Salmonella is also increasing. This increase is not only in the percentage isolates resistant to a particular antibiotic, but also the development of resistance against newer antibiotics. The increased occurrence of integrons is particularly worrying. Integrons can harbour a varying set of antibiotic resistance encoding gene cassettes. Gene cassettes can be exchanged between integrons. Although the gene cassettes currently present in Salmonella integrons encode for older antibiotics (however, some still frequently used) gene cassettes encoding resistance against the newest antibiotics has been documented in Enterobacteriaceae. Furthermore, beta-lactamases with activity against broad-spectrum cephalosporins, which are often used in empiric therapy, have been found associated with integrons. So, empiric treatment of Salmonella infections becomes increasingly more difficult. The most worrisome finding is that virulence and resistance plasmids form cointegrates. These newly formed plasmids can be selected by antibiotic pressure and thereby for virulence factors. Taken together these trends may lead to more virulent and antibiotic-resistant Salmonella.     

Antigenic polysaccharides of bacteria: 42. Structures of O-polysaccharides from two Shigella dysenteriae type 8 strains

The structure of the O-polysaccharide (O-antigen) from Shigella dysenteriae type 8 bacteria (strain 599) was corrected using modern NMR techniques (structure 1). The revisions concerned the position of the Glc residue (in the main, but not the side chain), the site of its substitution, and the configuration of the O-glycoside linkage of the GlcNAc residue. The S. dysenteriae type 8 bacterium (strain G1221), the second investigated representative, was found to produce another structural variant of the O-polysaccharide. It contains GlcNAc instead of the Glc residue in the main chain (structure 2). This data may lead to approval of division of S. dysenteriae type 8 into two subtypes: