肺炎链球菌荚膜多糖纯化及质量控制方法的研究现状

肺炎链球菌(简称肺炎球菌)荚膜多糖(capsular polysaccharide,CPS)位于肺炎球菌的最外层,是肺炎球菌主要的毒力因子之一,也是有效的保护性抗原.获得高质量的肺炎球菌CPS,是肺炎球菌多糖疫苗(pneumococcal pol-ysaccharide vaccine,PPV)和肺炎球菌结合疫苗(pn...     

不同剂量肺炎链球菌荚膜多糖与蛋白结合物的免疫原性比较

用肺炎链球菌19F型、23F型的荚膜多糖(C-PS)分别和破伤风类毒素(TT)蛋白结合,制备了两个型别的多糖-蛋白结合物(19F-TT,23F-TT)。为探讨结合物的最适免疫剂量,以10μg多糖和含1μg、3μg、9μg、27μg多糖的结合物经腹腔免疫NIH小鼠,ELISA方法检测小鼠血清中多糖和含不同多糖的结合物所产生的特异性IgG抗体。结果在不同的剂量免疫小鼠后,3μg剂量组在免疫三针后可诱生高浓度的抗体,但随着免疫剂量的增加,9μg与27μg诱生的抗体浓度较3μg诱生的抗体浓度之间并无显著性差异。含多糖3μg的19F型和23F型多糖蛋白结合物剂量组可诱生高浓度的特异性IgG抗体。     

肺炎链球菌5型发酵工艺的优化

目的:采用正交试验设计方法进行肺炎链球菌5型发酵工艺的研究。方法:根据正交试验设计表L9(34)设计的试验条件组合进行了9次肺炎链球菌5型的发酵,采用70升发酵罐进行发酵工艺的摸索,提取了肺炎链球菌5型荚膜多糖粗糖。结果:最佳的发酵培养条件组合为温度37℃、葡萄糖20克/升、大豆胨15克/升、pH值7.3,最佳的纯化条件组合为冷酚抽提三次、沉核酸乙醇浓度23%、超滤膜孔径50kD、最终沉糖乙醇浓度60%,在此筛选得到的最佳条件下,连续进行了5个批次肺炎链球菌5型的发酵与荚膜多糖提取,荚膜多糖粗糖的平均收率为808.6mg/L,相对标准偏差为3.84%。结论:上述发酵培养条件组合适合用于肺炎多糖疫苗的研究和生产。     

肺炎链球菌糖代谢蛋白CcpA对荚膜多糖的调控作用

摘要:【目的】研究肺炎链球菌糖代谢蛋白CcpA对肺炎链球菌荚膜多糖(CPS)的调控作用。【方法】利用大肠杆菌(Escherichia coli)BL21(DE3)工程菌原核表达CcpA蛋白,使用Ni2+亲和层析的方法纯化蛋白。利用纯化后的CcpA蛋白免疫昆明小鼠并制备多克隆抗体;采用ELISA法测定抗CcpA抗体效价。随后,利用Westertn blot方法分析CcpA蛋白在肺炎链球菌中的保守性。另外,利用EMSA方法分析CcpA与cps基因座启动子区域片段的结合。最后,构建ccpA基因缺失株和ccpA基因回复株;利用ELISA法测定野生D39菌 株、ccpA基因缺失株和ccpA基因回复株的荚膜多糖含量。【结果】Western blot结果显示CcpA蛋白在多种血清型的肺炎链球菌均有表达,CcpA蛋白可与cps基因座启动子区域结合,且呈剂量依赖性;ccpA基因缺失时,细菌CPS含量升高,回复表达CcpA蛋白后,CPS含量显著降低。【结论】CcpA是肺炎链球菌中一种保守表达的蛋白,可通过调节cps基因座启动子负性调控肺炎链球菌荚膜多糖的表达。     

肺炎链球菌SpxA蛋白的原核表达、纯化及多克隆抗体的制备

目的 构建肺炎链球菌SpxA蛋白的原核表达系统,制备其多克隆抗体.方法 设计引物,利用PCR技术扩增肺炎链球菌D39菌株的spxA基因,并插入表达载体pET-28a（＋）内,测序鉴定.重组质粒转化至大肠埃希菌BL21（DE3）中,以IPTG诱导表达含6个组氨酸标签的SpxA重组蛋白,经Ni-NTA亲和层析柱纯化后,以其为抗原免疫BALB/c小鼠制备多克隆抗体.用ELISA及Western印迹方法分别检测多克隆抗体的效价及特异性.结果 从大肠埃希菌中诱导出高表达的SpxA重组蛋白,纯化后免疫小鼠获得抗血清,ELISA测定其效价可达1：2 560 000以上,Western印迹结果显示其能特异性地作用于肺炎链球菌SpxA.结论 成功构建了pET-28a（＋）-spxA原核表达质粒,获得了高纯度的目的 蛋白和高滴度、高特异性的多克隆抗体.     

肺炎链球菌假想蛋白SPD0873的表达纯化及保守性分析

目的通过构建原核表达载体,获得纯化的肺炎链球菌S．pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S．pn菌株中的保守性。方法分离培养D39型肺炎链球菌,获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32（a）内,测序鉴定。将重组质粒转化到E．coli Rossetta（DE3）中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni—NTA树脂纯化后,获得的重组蛋白用SDS—PAGE和Western印迹鉴定;将鉴定后纯化的蛋白免疫BALB／C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达。纯化蛋白免疫BALB／C小鼠获得高滴度、高特异性的的多克隆抗体,Western印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达。结论成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。     

合成肺炎链球菌荚膜多糖基因的研究进展

肺炎链球菌是导致婴幼儿和老年人罹患肺炎、脑膜炎、中耳炎等疾病的主要病原体之一,其致病力与位于细菌表面的荚膜多糖密切相关,而荚膜多糖层的薄厚和多糖结构是影响致病力的主要因素。在分子水平探索参与荚膜多糖合成的相关基因,不仅有助于进一步理解肺炎链球菌的致病机理,而且可从基因水平选育高表达荚膜多糖的肺炎链球菌菌株用于多糖疫苗的研发。鉴于此,现就合成肺炎链球菌荚膜多糖基因的作用机制和研究方法作一综述。     

一种溶菌酶样蛋白对肺炎链球菌毒力及荚膜多糖合成的影响

摘要：【目的】为了研究肺炎链球菌（Streptococcus pneumoniae, S.pn）的一种假想的溶菌酶样蛋白在细菌生物学性状及其致病中的作用。【方法】利用长臂同源PCR对该基因进行敲出,并同时构建带有拯救质粒的缺失菌株,观察D39野生菌、缺失菌与带有拯救质粒的缺失菌株在相关生物学性状及其致病力改变,从而鉴定这种假想溶菌酶样蛋白的功能。【结果】缺失菌与野生菌相比,细菌生长减缓,毒力下降,荚膜多糖合成明显减少。而将拯救质粒转入缺失菌株后,该溶菌酶样蛋白的mRNA表达水平较野生菌高,其毒力及荚膜合成     

~1H-NMR用于肺炎链球菌荚膜多糖质量控制的尝试

纯化的6B、18C血清型肺炎链球菌荚膜多糖用生化试验和免疫学试验检测分析后再用一维氢谱核磁共振波谱(1H-NMR)法分析。生化检测其相应多糖的主要化学基团含量是否合格,免疫学检测旨在了解多糖纯化工艺是否影响了多糖的抗原活性,并间接佐证纯化多糖的生化特性是否正确。在此基础上进行1H-NMR分析,可以对纯化多糖的特性有进一步的了解。结果表明,常规的生化检测试验和免疫学检测试验并联合应用1H-NMR分析法后可更好地控制纯化肺炎链球菌荚膜多糖的质量。     

肺炎链球菌性肺炎诊断方法研究进展

肺炎链球菌是一种常见的细菌性肺炎的病原体,快速、准确的确定病原体是控制疾病的关键。本文就目前诊断肺炎链球菌性肺炎的免疫层析法、酶联免疫吸附法、胶乳颗粒凝集试验等研究进展作以简要综述。     

Synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 14

Synthesis of the regular branched polysaccharide [-6(Gal beta 1-4)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-]n structurally corresponding to capsular polysaccharide of Streptococcus pneumoniae type 14 involves blockwise synthesis of a tritylated 1,2-O-(1-cyano)ethylidene tetrasaccharide derivative from lactosamine and lactose precursors followed by stereospecific polycondensation of the tetrasaccharide monomer.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Monoclonal antibodies with specificities for Streptococcus pneumoniae group 9 capsular polysaccharides

Streptococcus pneumoniae group 9 includes four capsular polysaccharide types: 9A, 9L, 9N and 9V. We have generated four mouse monoclonal antibodies against group 9 polysaccharide using heat-treated S. pneumoniae strains of different capsular polysaccharides types as immunogens. The specificities of the monoclonal antibodies were determined by ELISA using capsular polysaccharide directly coated to the wells as antigens and by dot blotting with heat-treated bacteria. Two groups of monoclonal antibodies were found. The first group included two monoclonal antibodies which were found to be capsular type specific. The second group was monoclonal antibodies that bound to epitopes shared by two or three pneumococcal group 9 types. The monoclonal antibody 204,A-4 (IgM) was found to be specific for S. pneumoniae type 9N. The binding of the type 9V specific monoclonal antibody 206,F-5 (IgG1) was found to be dependent upon O-acetyl groups. Monoclonal antibody 205,F-3 (IgM) reacted also with type 9V, but was found to cross-react with types 9A and 9L. The binding of this monoclonal antibody to polysaccharide 9V was not dependent upon O-acetyl moieties. The fourth monoclonal antibody (214,G-5, isotype IgM) did not show any correlation between reactivity with isolated polysaccharides and dot blotting with relevant bacteria. The monoclonal antibody reacted with polysaccharides 9A and 9L in ELISA, but not with the homologous bacteria.     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 5

The structure of the capsular polysaccharide (S5) elaborated by Streptococcus pneumoniae type 5 has been investigated by using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure: (Formula: see text) In this structure, L-PneNAc stands for 2-acetamido-2,6-dideoxy-L-talose (pneumosamine) and D-Sug for 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose. The latter sugar accounts for the lability of S5 towards alkali. N.m.r. spectra indicate heterogeneity in S5, most probably associated with the hexosyl-4-ulose residue.     

Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 7A

Application of methylation analysis, specific degradations, and n.m.r. spectroscopy to the capsular polysaccharide elaborated by Streptococcus pneumoniae type 7A indicates a hexasaccharide repeating-unit with the structure (Formula; see text).     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 18F

The structure of the capsular polysaccharide elaborated by Streptococcus pneumoniae type 18F (S18F) has been investigated by using n.m.r. spectroscopy, methylation analysis, and characterisation of oligosaccharides obtained on partial hydrolysis. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure. (formula; see text) In this structure, the absolute configuration of the glycerol phosphate moiety has not been determined, but is assumed to be D-glycerol 1-phosphate (sn-glycerol 3-phosphate). The location of an O-acetyl group at O-6 of the terminal alpha-D-glucopyranosyl groups is tentative only.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 18A

The structure of the capsular polysaccharide (S18A) elaborated by Streptococcus pneumoniae type 18A has been investigated by using methylation analysis and n.m.r. spectroscopy. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure. (formula; see text) In this structure, the absolute configuration of the glycerol 1-phosphate moiety has not been determined but is assumed to be D from biosynthesis considerations. The structure of S18A is, as expected, closely similar to those determined for S18F and S18C.