沙门氏菌属鞭毛蛋白共同抗原表位的初步定位

间接免疫荧光试验和免疫胶体金试验中，证实ＣＢ８、ｄｅ７、ｃｃ６、ＤＤ４识别的抗原表位重复出现于鞭毛蛋白上，单抗与已知菌株的反应特性显示，４株单抗识别的抗原表位不同，表明鞭毛蛋白的属共同抗原具有多种表位的特性。用上述沙门氏菌鞭毛蛋白共同抗原单抗测定证实鞭毛蛋白共同抗原表位广泛分布于属内，而在属外出现的频率很低，且不受鞭毛位相改变的影响，表现出其属特异的保守性，这些特性不同于常规Ｏ、Ｈ抗原分布特征。     

4株沙门氏菌鞭毛蛋白属共同抗原组分免疫原性及免疫保护性的研究

采用酸解法、单抗亲和层析提纯了鼠伤寒沙门氏菌（Ｓ．ｔｙｐｈｉｍｕｒｉｕｍ）鞭毛蛋白Ｈｉ。经电镜和ＳＤＳＰＡＧＥ鉴定，发现４２ｋＤ的蛋白带，即鞭毛蛋白（Ｆｌａｇｅｌｌｉｎ）。它能够刺激小鼠产生特异性抗体，酶联免疫吸附试验（ＥＬＩＳＡ）、凝集试验（ＤＡ）测定其Ｈｉ抗体效价分别为１：１０４、１：１０３。用其他非Ｈｉ抗原的沙门氏菌包板，ＥＬＩＳＡ效价可达１：１００～１：１０００，ＤＡ阴性。这表明沙门氏菌鞭毛蛋白上存在着属特异共同抗原表位，且免疫原性较好，该类表位免疫性比分型Ｈ抗原弱，提示沙门氏菌分型Ｈ抗原表位是鞭毛蛋白分子上的优势表位。用沙门氏菌鞭毛蛋白属特异共同抗原表位单抗ｄｅ７研究被动保护作用，结果显示该表位被动保护作用很小。本研究为沙门氏菌鞭毛蛋白抗原多样性及特性研究提供了新的认识，并为临床上诊断沙门氏菌感染提供了新的检测对象。     

单克隆抗体测定沙门氏菌鞭毛蛋白属共同抗原表位的分布特性

本研究应用抗沙门氏菌鞭毛蛋白共同抗原的４株单克隆抗体所建立的直接ＥＬＩＳＡ法，测定了该类抗原表位在沙门氏菌属内外的分布特征。对１８４株覆盖Ａ至Ｏ６７血清群沙门氏菌，９６株其他肠道杆菌和８株革兰氏阳性菌的测定结果显示，该共同抗原表位广泛分布一门氏菌金属内，而在属外出现的频率很低。这类表位既存在Ｉ相鞭毛，也存在于Ⅱ相鞭毛。它们的上述分布特性不同分型抗原Ｏ、Ｈ的分布特征。由此可见，沙门氏菌鞭毛蛋白共同抗     

单克隆抗体测定沙门氏菌鞭毛蛋白属共同抗原表位的分布特性

本研究应用抗沙门氏菌鞭毛蛋白共同抗原的４株羊克隆抗体所建立的直接ＥＬＩＳＡ法，测定了该类抗原表位在沙门氏菌属内外的分布特征。对１８４株覆盖Ａ至Ｏ６７血清群沙门氏菌、９６株其他肠道杆菌和８株革兰氏阳性菌的测定结果显示，该共同抗原表位广泛分布于沙门氏菌全属内，而在属外出现的频率很低。这类表位既存在于Ⅰ相较毛，也存在于Ⅱ相鞭毛。它们的上述分布特性不同于分型抗原Ｏ、Ｈ的分布特征。由此可见，沙门氏菌鞭毛蛋白共同抗原表位具有高度的属特异性，可作为该菌属识别标志，在沙门氏菌快速检验和抗原结构研究方面具有重要应用价值。     

人巨细胞病毒单克隆抗体的研制检定和应用

采用人巨细胞病毒（ＨＣＭＶ）ＡＤ１６９株作为免疫原，制备出１３株鼠－鼠杂交瘤细胞系。对其中的６株进行了检定．免疫印迹试验结果表明：单克隆抗体（ＭｃＡｂ）７Ｂ４、７Ｄ７、７Ｅ１１、８Ｅ８和８Ｄ６相对应的ＨＣＭＶ多肽分子量分别为４６、１５０、３８、５１７２和６５ｋＤ．ＨＣＭＶ感染人胚肺二倍体细胞（２ＢＳ）后不同时间制成抗原片，与ＭｃＡｂ作间接免疫荧光试验。结果表明：ＭｃＡｂ８Ｂ８相应的病毒多肽为即刻早期抗原，其它５株ＭｃＡｂ相应的病毒多肽均为晚期抗原，６株ＭｃＡｂ等量混合后，标上辣根过氧化物酶，用于ＩｇＭ抗体捕获法ＥＬＩＳＡ（ＭａｃＥＬＩＳＡ）中，并与间接ＥＬＩＳＡ（ＩＥＬＩＳＡ）同时检测ＨＣＭＶ－ＩｇＭ．在未经选择的１００份脐带血中，两法均为阳性的３份，两法均为阴性的９４份；ＭａｃＥＬＩＳＡ阳性而ＩＥＬＩＳＡ阴性的２份血清的特异性试验证明，ＨＣＭＶ－ＩｇＭ确为阳性．ＩＥＬＩＳＡ阳性而ＭａｃＥＬＩＳＡ阴性的１份血清的特异性试验证明，它是由ＲＦ引起的假阳性。     

编码与大鼠ERα-甘露糖苷酶同源的人6A8 cDNA在真核细胞的表达

目的观察６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞的表达。方法Ｎｏｒｔｈｅｒｎｂｌｏｔ，构建６Ａ８ｃＤＮＡ表达载体ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ，转染ＣＯＳ－７细胞，及基因免疫小鼠。结果６Ａ８ｃＤＮＡ有４．３ｋｂ和３．５ｋｂ两种ｍＲＮＡ转录形式。４．３ｋｂｍＲＮＡ以外周血白细胞最丰富，其次为淋巴结、脾脏和骨髓，胸腺组织表达较少，胎肝则缺如。３．５ｋｂ者也以外周血白细胞最丰富，其次为肝脏、淋巴结和骨髓，胸腺和脾脏表达量最少。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ在ＣＯＳ－７细胞表达了分子量４５０００左右的蛋白质，与单抗６Ａ８有反应。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ基因免疫小鼠，６／１０只小鼠血清与人活化Ｂ细胞株３Ｄ５细胞膜有反应，５／５只对照小鼠血清呈阴性反应。结论６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞获得表达，但此ｃＤＮＡ非为全长。     

抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体的研制及 …

本文采用杂交瘤技术，制备了７株抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体的杂交瘤细胞株。对３株ｍＡｂ（２Ｃ８，２Ｅ６和５Ｄ７０进行了鉴定，ｍＡｂ ２Ｃ８和５Ｄ７为ＩｇＭ，２Ｅ６为ＩｇＧ１亚类，可分别识别不同的抗原表位，均具有很强的特异性，即只与甲型副伤寒沙门氏菌及其鞭毛抗原起反应，而与相关肠道细菌：尼亚里木沙门氏菌，达卡沙门氏菌及伤寒沙门氏菌，乙型和丙型副伤寒杆菌，猪霍乱沙门氏菌，纽波特沙门氏菌等均无     

我国急性散发性戊型肝炎病毒部分核苷酸序列分析

用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）自深圳、长春、杭州等地４１份急性散发性戊型肝炎病人血清中获得２８株ＨＥＶｃＤＮＡ，对其中３株ＨＥＶｃＤＮＡ的ＯＲＦ２基因片段，用荧光法直接测定其核苷酸序列，并与戊型肝炎病毒墨西哥株（Ｍ）、缅甸株（Ｂ）和新疆流行株（ＣＨ１·１）进行了比较，结果该３株散发性ＨＥＶ与Ｍ株的核苷酸序列同源性分别为８０．２％、７９．９％和７９．４％；与Ｂ流行株的同源性为９５．５％、９３．９％和９５．１％；与散发株的同源性为９３．４％、９２．３％和９３．８％；与ＣＨ１·１的同源性为９７．０％、９６．５％和９５．９；表明该３株散发性ＨＥＶ与ＨＥＶ（Ｂ）和ＣＨ１·１的核酸序列同源性较高，可能属同一亚型。     

人心肌肌钙蛋白T单克隆抗体的研制及鉴定

以人心肌肌钙蛋白Ｔ（ｃＴｎＴ）为抗原，采用脾内免疫法，免疫ＢＡＬＢ／ｃ小鼠，取其脾淋巴细胞与小鼠Ｓｐ２／０细胞融合，经间接ＥＬＩＳＡ法筛选，三次克隆化后获得５株能稳定分泌抗ｃＴｎＴ单克隆抗体（ＭｃＡｂ）的杂交瘤细胞Ｇ３、Ｇ８、Ｇ１０、Ａ５和Ａ７。免疫球蛋白亚类鉴定其中１株为ＩｇＧ２ａ，４株为ＩｇＭ。染色体数目９２～１１０条。将Ｇ３、Ｇ８、Ｇ１０、Ａ５的单克隆抗体腹水做１：１００稀释与ＬＤＨ、ＣＫ、ＣＫＭＢ和ＧＯＴ等心肌酶均无交叉反应。５株ＭｃＡｂ的腹水效价为３．２×１０－６～１．６×１０－７。ＭｃＡｂ相加试验表明，Ａ５和Ｇ３可识别不同的抗原表位。     

狼疮性肾炎患者淋巴细胞亚群比率和免疫球蛋白水平的变化

目的 探讨狼疮性肾炎（ＬＮ）患者的淋巴细胞亚群和免疫球蛋白（Ｉｇ）的变化及其意义。方法采用淋巴细胞的膜抗原双标记染色法及ＥＬＩＳＡ，对６０例ＬＮ患者的细胞与体液免疫功能进行前瞻性研究。结果①与对照组相比较，活动期ＬＮ患者的ＣＤ３＋ＣＤ４＋细胞的比率（Ａ组：伴肾病综合征者）为（ｌ６．３ ±７．９）％，Ｂ组（不伴肾病综合征者）为（２０．８±１０．１）％］和ＣＤ１６＋＋ＣＤ５６＋细胞的比率［Ａ组为（８．６±５．７）％，Ｂ组为（１５．２±６．２）％明显减少；ＣＤ３+ ＣＤ8+”细胞的比率［Ａ组为（４８．３±１０．７）％，Ｂ组为（３５．９±９．８）％］明显增高；ＣＤ３+ ＣＤ４+ ／ＣＤ３+ ＣＤ８＋细胞的比值＜１（Ａ组为０．４３±０．２１，Ｂ组为０．８７±０．２５），且Ａ组与８组相比较差异明显（Ｐ＜０．０５）。②与活动期ＬＮ患者相比较，治疗后处于稳定期的 ＬＮ患者 ＣＤ３＋ＣＤ４＋细胞的比率卜组为（２８．８±８．ｌ）％，Ｂ组为（３２．８± ７．ｌ）％］和 ＣＤ１６＋＋ＣＤ５６＋细胞的比率［Ａ组为（１８．９ ± １２．５）％，Ｂ组为（２４．０±８． ９）％，以及 ＣＤ３＋ＣＤ４＋／ＣＤ３＋ＣＤ８＋细胞的比值（Ａ组为 ０．９７±２．３，Ｂ组     

Host and bacterial factors affecting induction of immune responses to flagellin expressed by attenuated Salmonella vaccine strains

Previous observations demonstrated that the delivery of recombinant Salmonella enterica serovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either type d flagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuated Salmonella strains. The reduced anti-type d flagellin antibody responses in mice immunized via mucosal routes with three doses of flagellated S. enterica serovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses to Salmonella flagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized with S. enterica serovar Typhimurium strains developed anti-type i flagellin sera and secreted antibody responses, which indicated that the serovar of the Salmonella vaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellated S. enterica serovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated type d flagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-gamma) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC), in mice orally immunized with a Salmonella vaccine strain expressing hybrid flagella. These results indicate that the immunogenicities of Salmonella flagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-gamma production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinant S. enterica serovar Dublin vaccine strains.     

Molecular characterization of Salmonella enterica subsp. enterica serovar choleraesuis field isolates and differentiation from homologous live vaccine strains suisaloral and SC-54.

Four independent molecular methods were used to characterize the Salmonella enterica subsp. enterica serovar choleraesuis live vaccine strains SC-54 and Suisaloral and to differentiate them from S. choleraesuis field isolates. Plasmid analysis revealed the presence of seven plasmid profiles. A virulence plasmid of 52-kbp was identified by hybridization with an spvB-spvC gene probe in each of the S. choleraesuis field isolates and in the Suisaloral vaccine strain, but not in the SC-54 vaccine strain. Ribotyping, performed with a gene probe that recognized 23S, 16S, and 5S rRNA genes, resulted in three closely related hybridization patterns. IS200 elements were not detected in the field isolates or in the two S. choleraesuis live vaccine strains. Macrorestriction analysis with the enzymes XbaI, SpeI, NotI, and SfiI differentiated the 29 S. choleraesuis strains included in this study into 10, 13, 8, and 13 different fragment patterns, respectively. While the Suisaloral vaccine strain showed a unique XbaI macrorestriction pattern, the fragment patterns of the SC-54 strain obtained with the different enzymes were shared by 2 to 18 S. choleraesuis field strains. A combination of plasmid analysis and macrorestriction analysis proved to be most suitable for the molecular typing of S. choleraesuis and the differentiation of both live vaccine strains from field isolates of this serovar.     

单克隆抗体在沙门氏菌血清学鉴定中的应用研究

应用肠杆菌科共同抗原单抗AA9、沙门氏菌属特异单抗试剂(CB_8+de7)、沙门氏菌O、H抗原单抗对1271株菌株进行鉴定,试验结果表明,AA9和CB_8+de7可用于沙门氏菌科、属的初步鉴定;沙门氏菌O、H抗原单抗只能选择性地与具有相应抗原的菌株的发生反应,而不与其它菌株产生交叉反应,其敏感性和特异性优于常规血清,完全可以取代后者用于沙门氏菌血清群、型的鉴定。可见,沙门氏菌单抗的应用为沙门氏菌的鉴定提供了新方法,文中进一步讨论了上述单抗的应用前景。     

CD4+-T-cell responses generated during murine Salmonella enterica serovar Typhimurium infection are directed towards multiple epitopes within the natural antigen FliC

The flagellar filament protein FliC is a natural antigen recognized by memory CD4+ T cells recovered from Salmonella enterica serovar Typhimurium-infected humans and mice. To further investigate T-cell responses to FliC, we derived FliC-specific CD4+-T-cell clones from mice of two different haplotypes following oral S. enterica serovar Typhimurium infection. Using C-terminal truncations of MalE-FliC recombinant fusion proteins, we mapped antigenic activity to four different regions of FliC; three of the four epitope-containing regions were present in both FliC and the alternate flagellin subunit FljB. We determined that two novel FliC epitopes were also present in flagellins from several gram-negative enteric bacterial species: E(k)-restricted FliC 80-94 (amino acids 80 to 94) and A(b)-restricted FliC 455-469. Further mapping confirmed the presence of two previously identified FliC epitopes: A(k)-restricted FliC 339-350 and A(b)-restricted FliC 428-442. Therefore, like the recognition site of the innate immune receptor Toll-like receptor 5, three of four FliC epitopes recognized by CD4+ T cells colocalize in the D0/D1 domains of FliC. Salmonella-infected macrophages and dendritic cells stimulated epitope-specific CD4+-T-cell proliferation; infected dendritic cells also activated T cells to produce gamma interferon. These data demonstrate that Salmonella infection generates murine CD4+-T-cell responses to multiple epitopes in the natural antigen FliC and that recognition of infected phagocytes by FliC-specific CD4+ T cells triggers effector functions known to be essential for protective immunity. Together, these data suggest that FliC-specific CD4+ T cells may contribute to cell-mediated host defenses against Salmonella.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.     

Characterization of Salmonella serovars by PCR-single-strand conformation polymorphism analysis

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.     

实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。     

Development of a multiplex PCR technique for detection and epidemiological typing of salmonella in human clinical samples

We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:-. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.     

Simultaneous analysis of multiple enzymes increases accuracy of pulsed-field gel electrophoresis in assigning genetic relationships among homogeneous Salmonella strains

Due to a highly homogeneous genetic composition, the subtyping of Salmonella enterica serovar Enteritidis strains to an epidemiologically relevant level remains intangible for pulsed-field gel electrophoresis (PFGE). We reported previously on a highly discriminatory PFGE-based subtyping scheme for S. enterica serovar Enteritidis that relies on a single combined cluster analysis of multiple restriction enzymes. However, the ability of a subtyping method to correctly infer genetic relatedness among outbreak strains is also essential for effective molecular epidemiological traceback. In this study, genetic and phylogenetic analyses were performed to assess whether concatenated enzyme methods can cluster closely related salmonellae into epidemiologically relevant hierarchies. PFGE profiles were generated by use of six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for 74 strains each of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium. Correlation analysis of Dice similarity coefficients for all pairwise strain comparisons underscored the importance of combining multiple enzymes for the accurate assignment of genetic relatedness among Salmonella strains. The mean correlation increased from 81% and 41% for single-enzyme PFGE up to 99% and 96% for five-enzyme combined PFGE for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains, respectively. Data regressions approached 100% correlation among Dice similarities for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains when a minimum of six enzymes were concatenated. Phylogenetic congruence measures singled out XbaI, BlnI, SfiI, and PacI as most concordant for S. enterica serovar Enteritidis, while XbaI, BlnI, and SpeI were most concordant among S. enterica serovar Typhimurium strains. Together, these data indicate that PFGE coupled with sufficient enzyme numbers and combinations is capable of discerning accurate genetic relationships among Salmonella serovars comprising highly homogeneous strain complexes.