福氏2a痢疾杆菌O—特异性多糖与破伤风类毒素结合疫苗的制备及其免疫学特性

通过己二酸二肼（ADH）将福氏2a痢疾杆菌脂多糖（LPS）经酸水解脱毒纯化后得到的O－SP和破伤风类毒素（TT）蛋白共价结合,制备了福氏2a痢疾杆菌结合物。以2.5μg多糖或含2.5μg多糖的结合物经皮下注射免疫NIH品系雌性小鼠,同时以25μg多糖或含25μg多糖的结合物经耳缘静脉注射家兔。用ELISA方法分别检测小鼠和家兔血清中抗脂多糖抗体水平及抗TT水平,并用豚鼠血清补体介导进行了体外杀菌实验。结果显示：用本实验方法提取的多糖,合成的多糖衍生物,多糖－蛋白结合物都具有福氏2a痢疾杆菌O－抗原特异性,其化学组成及结构特异性和国外文献报道基本一致。单独用多糖免疫小鼠和家兔未能诱导LPS抗体,而结合物免疫小鼠和家兔的血清诱导出了较高的抗LPS IgG抗体及抗TT抗体,并有较强的体外杀菌活性。产生的抗体存在着免疫记忆性,可以产生再次免疫应答加强反应。     

志贺氏Ⅰ型痢疾杆菌O—特异性多糖与破伤风类毒素结合疫苗 …

从志贺氏１型痢疾村菌ＬＰＳ中分离纯化出料捩的,以ＡＤＨ为连接剂将其与ＴＴ结合形成Ｏ－ＳＰ－ＴＴ结合疫苗,并用此结合疫苗免疫ＮＩＨ小鼠,结果显示使用Ｏ－ＳＰ免疫后,小鼠血清中没有抗ＬＰＳ抗体产生,而用Ｌ－ＳＰ－ＴＴ免疫后鼠血清中生了抗ＬＰＳＩｇＧ和ＩｇＭ抗体,且ＩｇＧ抗体水平高于ＩｇＭ抗体;Ｏ－ＳＰ－ＴＴ免疫组等二次和第三次免疫后ＩｇＧ我有显著的升高（Ｐ〈０．０１）,但第二次和第三次免疫后血清ＩｇＭ     

甲型副伤寒菌O-特异性多糖与破伤风类毒素结合菌苗的制备及其在小鼠体内的免疫原性

以脱毒后去除类脂A的甲型副伤寒杆菌高分子量O SP1和低分子量O SP2 为特异性抗原 ,以破伤风类毒素 (TT)为蛋白质载体 ,用己二酸二肼 (ADH)作为连接剂制备的两种结合物及其多糖免疫NIH小鼠 ,结果显示单独注射O SP1或O SP2 免疫小鼠后 ,均不能刺激小鼠产生抗 LPS抗体 ;而用O SP1 TT和O SP2 TT结合物免疫后 ,小鼠血清中均产生了特异性抗 LPSIgG和IgM抗体 ,且O SP1 TT免疫组血清中IgG抗体水平明显高于O SP2 TT免疫组 ,两组结合物免疫血清中抗体类型以IgG为主。O SP1 TT结合物免疫组第二次免疫和第三次免疫后IgG抗体水平均较前一次有显著升高 (p<0 0 1)说明 ,O SP1 TT结合物具有加强应答效果。补体介导的体外杀菌试验证明 ,O SP1 TT与O SP2 TT结合物免疫血清具有特异性杀菌活性。     

铜绿假单胞菌6型O-特异性多糖与破伤风类毒素结合物免疫原性研究

目的研制有效防治铜绿假单胞菌感染的疫苗。方法用热酚水法提取铜绿假单胞菌6型(IATS 6)菌株的脂多糖(LPS),去除类脂A并纯化O-特异性多糖(O-SP),用CDAP活化O-SP,己二酸二肼作连接臂,在EDAC作用下,与破伤风类毒素结合制备出O-SP-TT结合物并对该结合物进行小鼠免疫原性试验和免疫保护性试验。结果制备的结合物在小鼠免疫原性试验中,产生了高效价的IgG抗体,与O-SP试验组相比,差异具有统计学意义(P<0.05);免疫保护性试验表明,结合物免疫小鼠能很好的保护5～10 LD50活菌的腹腔攻击。结论该结合物有望成为防治IATS 6型铜绿假单胞菌感染的有效疫苗。     

宋内氏痢疾杆菌O-SP-TT结合疫苗的制备及其免疫学特性的研究

宋内氏痢疾杆菌（S.Sonnei)的一个重要保护性抗原是菌体抗原（O抗原）即细菌细胞壁中的脂多糖（LPS）成分,已证实血清中足够水平的抗LPS IgG抗体抗体即能对该菌提供免疫保护作用。本文选用Westphal热酚法提纯宋内氏痢疾菌LPS,经酸水解法进一步脱毒处理后得到无毒但却具弱免疫原性的O-特异性多糖（O－SP）,然后采用化学方法即通过连接剂己二酸二肼（ADH）将其共价结合到破伤风类毒素（TT）上,共制备得到了三批宋内氏痢疾杆菌O－SP－TT结合疫苗。经生化和免疫学方法检测证实我们提纯的LPOS,OSP及其合成的L－SP－AH衍生物,O－SP－TT结合物均具有宋内氏痢疾杆菌O抗原特异性,同时其核酸和杂蛋白质含量低（≤2%),表明纯度较高。同时经小鼠免疫原性试验证实三批结合物免疫小鼠后产生的抗LPS IgG抗体滴度均比单一O－SP免疫后产生的要高出10倍以上,且结合物再次注射后存在加强应答。补体介导的体外杀菌力试验表明结合物免疫小鼠后诱导产生的血清抗LPSIgG抗体对宋内氏痢疾杆菌具特异性杀菌活性。本文结果表明宋内氏痢疾杆菌O-SP-TT结合疫苗可望作为一种有效的侯选痢疾疫苗做进一步的大规模人体观察。     

C群奈瑟氏脑膜炎球菌荚膜多糖-TT结合疫苗的制备及其在小鼠体内免疫原性的研究

将C群脑膜炎球菌荚膜多糖以ADH作为间隔剂与TT结合形成GCMP-TT结合疫苗,然后用此结合疫苗免疫NIH小鼠,结果显示使用GCMP免疫小鼠后仅能产生较低水平抗GCMP的IgG抗体,而用GCMP-TT免疫后小鼠血清中产生了较GCMP免疫显著增高抗GCMP的IgG抗体,并且GCMP-TT组第二次和第三次免疫后与初次免疫相比,IgG抗体水平均有显著升高(P<0.01),表明GCMP-TT结合疫苗具有免疫记忆和再次免疫加强应答效应。补体介导的血清抗体体外杀菌试验结果证明,GCMP-TT结合疫苗组免疫小鼠诱导的抗体IgG比GCMP组具有增强的体外杀菌活性。     

14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗的研制

将14型肺炎球菌的荚膜多糖(PS)与破伤风类毒素(TT)通过化学方法结合,制备成多糖-蛋白结合疫苗(PS14-TT)。用该结合疫苗免疫小鼠,在小鼠体内产生了高滴度的PS-IgG抗体和TT-IgG抗体,且再次注射后有加强应答效应,表明制备的结合疫苗保留了完好的抗原性,具有胸腺依赖性抗原的特性。     

A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究

采用溴化氰(CNBr)活化多糖,以无水己二酸二肼(ADH)作为连接剂,1乙基13(3二甲基氨基丙基)碳化二亚胺(EDAC)为偶联剂制备A群奈瑟氏脑膜炎球菌荚膜多糖(GAMP)与破伤风类毒素(TT)的结合物,经皮下免疫NIH小鼠,用ELISA检测小鼠血清中抗GAMP及抗载体蛋白的IgG抗体水平。用补体介导的体外杀菌试验检测血清中GAMP抗体的杀菌活性。结果显示,实验中制备的多糖衍生物和多糖蛋白质结合物都具有GAMP抗原特异活性。结合物免疫小鼠后可诱生比多糖单独免疫更高水平的GAMP血清IgG抗体,并能形成免疫记忆,产生再次应答。结合物免疫小鼠所诱生的血清GAMP抗体较之多糖组具有更强的体外杀菌活性。表明此方法制备的结合物可获得优于多糖的、稳定的特异免疫原性。     

b型流感嗜血杆菌结合疫苗中载体蛋白质的效力研究

考察b型流感嗜血杆菌(Hib)结合疫苗的载体蛋白质—破伤风类毒素(TT)的免疫原性,为百白破与Hib四联疫苗中TT的使用提供参考。方法:将小鼠随机分为四组,分别注射Hib结合疫苗、Hib与百白混合疫苗、Hib与百白破混合疫苗、破伤风类毒素,比较它们在NIH小鼠体内所诱导产生的特异性抗体水平,并对这四组疫苗进行破伤风效力保护试验。结果表明,破伤风类毒素组,Hib与百白破混合疫苗组刺激的TT抗体水平远大于Hib结合疫苗组和Hib与百白混合疫苗组。效力试验虽四组间没有差异,只说明一定的抗体量就可以对小鼠提供保护。认为Hib结合疫苗中的载体蛋白并不能替代百白破疫苗中的破伤风类毒素。     

肺炎链球菌18C型糖蛋白结合物的制备及其免疫原性

制备肺炎链球菌18C型荚膜多糖－破伤风类毒素结合物（CPS－TT）,测定结合物的理化性质,抗原特异性及其在动物中的免疫原性。结果显示,结合物能与相应的多糖和破伤风抗血清形成明显的沉淀线,蛋白／多糖比率为1．86,结合物分子大小（Kd值）为0．058。注射小鼠后可诱导明显的抗体应答,而且随着注射针次的增加,抗体反应水平明显增高,显示加强效应。结果表明,制备的肺炎链球菌糖蛋白结合物抗原性良好,具有胸腺依赖性的特性,在小鼠中显示较好的免疫原性。     

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy. The sequence of the sugar residues was determined using ¹H,¹H NOESY and ¹H,¹³C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structureSince small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.     

Circular dichroism of the O-specific polysaccharide of Vibrio cholerae O1 and some related derivatives

The O-specific polysaccharide (O-SP) of Vibrio cholerae O1 is a homopolymer of α-(1 → 2)-linked 4-amino-4,6-dideoxy- -mannopyranose whose amino group is acylated with 3-deoxy- -glycero-tetronic acid [

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. The circular dichroism (CD) of the O-SP as well as of a number of N-acyl (formyl, acetyl, 4-hydroxybutyl, 3-deoxy- and -glycero-tetronyl) derivatives of methyl α-glycosides of 4-amino-4,6-dideoxy- -mannopyranose (methyl α- -perosaminide) has been studied for solutions in water, acetonitrile and 1,1,1-trifluoroethanol. The strong solvent dependence of the sign and intensity of the CD observed for the monosaccharide amides bearing achiral acyl groups is explained by solvent-mediated change of the orientation of the amido group relative to the proximal hydroxyl group at C-3. A change in the population of the nonplanar conformers with a pyramidal arrangement of bonds at the amido nitrogen has also been considered. The effect of solvents upon the CD spectra of compounds bearing chiral N-acyl substituents is less pronounced than that of their counterparts bearing achiral N-acyl substituents. The sign of the CD for the O-SP was found negative in all solvents used. This result is in agreement with the negative sign of the CD of the n → π electron transition observed, independent of solvent, for the monosaccharide derivative containing the group, and the positive sign found for its -glycero-counterpart.     

The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strains

Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: [structure: see text]. NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.