铜绿假单胞菌外膜蛋白的免疫原性及免疫保护性

铜绿假单胞菌亦称绿脓杆菌,是一种最常见的条件致病菌。铜绿假单胞菌感染占院内感染的15 %以上,并还有上升趋势,这与铜绿假单胞菌对抗生素耐药率增高有关〔1〕。在国内外烧伤中心对革兰阴性细菌感染的临床调查表明,铜绿假单胞菌感染占首位(约5 0 %) 〔2〕。为此,人们一直在寻找有效的防治对策。既往研究的菌体疫苗缺点较多,如不同血清型保护率及保护时间不甚理想等。因此,发展高免疫保护性、低不良反应的亚单位疫苗非常必要。制备亚单位疫苗的关键在于找出与保护性免疫相关的抗原。细胞膜中的一种重要成分———外膜蛋白,在免疫方面的作用越…     

贝氏柯克斯体30kD外膜蛋白基因表达及免疫保护性的研究

贝氏柯克斯体可引起人类急、慢性Q热。急性Q热临床表现多种多样,主要症状为发热、头痛和肌肉酸痛,并常伴有肺炎、肝炎等。慢性Q热常发展为Q热性心内膜炎、骨髓炎。Q热疫苗是预防Q热流行的最有效手段,目前人用Q热疫苗是灭活的柯克斯体。虽然灭活疫苗的免疫保护效果很好,但注射该疫苗接种部位常出现不良反应。研究发现,在贝氏柯克斯体的Ⅰ相和Ⅱ相中含有相对分子质量(Mr)为30×10 3 的外膜蛋白,该蛋白具有良好的免疫原性和免疫反应性〔1〕。我们采用分子生物学技术,研制出贝氏柯克斯体Mr30×10 3 重组外膜蛋白,用重组蛋白免疫小鼠,对其免…     

中国莱姆病螺旋体PD91外膜蛋白A的克隆表达及免疫保护性的初步研究

目的：克隆并表达中国莱姆病螺旋体Borrelia garinii基因型代表菌株PD91的外膜蛋白A(OspA)，并对其免疫保护性进行 初步研究，为进一步研制莱姆病疫苗提供基础。方法：用聚合酶链反应（PCR）从莱姆病螺旋体PD91全基因组DNA中将OspA基因调出，插入原核表达载体P42，在大肠杆菌BI21(DE3)中表达，表达产物用SDS-PAGE、Western blot分析，并进行基因序列测定。用重组OspA（rOspA）免疫新西兰家兔，用间接免疫荧光（IFA）检测其血清特异性抗体（IgG），并进行体外中和试验，从而对其免疫保护性有初步的了解。结果：rOspA在宿主菌内表达高效、稳定；Western blot 显示其与抗OspA的多抗有较好的免疫反应性；用rOspA免疫新西兰家兔后，其血清抗体（IgG）效价显著升高（32倍），体外中和试验表明，每毫升兔抗rOspA血清可杀来源10^5个莱姆病螺旋体。结论：在国内首次成功地对中国莱妈病螺旋体Borrelia garinii基因型的OspA基因进行了克隆和表达。ROspA有较好的免疫保护性，可作为多价莱姆病疫苗的一种成分。     

骨髓瘤蛋白(IgDλ)独特型单克隆抗体的制备

本文用从1例骨髓瘤患者(IgDλ)尿中提取的λ链作为抗原,免疫Balb/c小鼠,通过两次融合,分别经3～4次克隆后获得86株分泌单克隆抗体(McAb)的杂交瘤细胞,经鉴定其中15株为抗独特型McAb。其中12株仅与同源性λ链和IgD反应,不与正常人λ链,k链,IgG、IgA、IgM、IgD、IgE及白蛋白(HSA)和一系列副球蛋白反应.间接免疫荧光法证明,抗独特型McAb与骨髓瘤患者外周血淋巴细胞和骨髓细胞阳性率高达23%,不与正常人群外周血淋巴细胞和骨髓细胞反应,其中部分McAb与实验室培养的浆细胞瘤株呈现阳性反应.     

肠出血性大肠杆菌O157:H7外膜蛋白A敲除菌株的构建及其黏附作用研究

目的利用Red同源重组技术构建肠出血性大肠杆菌(EHEC)O157∶H7外膜蛋白A(ompA)基因敲除菌株,研究敲除菌株的黏附能力变化,阐明OmpA在细菌黏附以及致病中的作用。方法利用Red同源重组技术对EHEC O157∶H7 ompA基因进行敲除,同时构建EHEC O157:H7 ompA回复突变菌株,最后通过细胞试验对EHEC O157∶H7野生型菌株、敲除菌株及回复突变菌株的黏附能力进行比较。结果成功构建了EHEC O157:H7 ompA基因敲除菌株及其回复突变菌株。细胞试验结果表明,与野生型菌株相比,敲除菌株的黏附能力明显下降,而将ompA基因进行回复突变后其黏附能力又得到回复。结论 OmpA在EHEC O157∶H7黏附HeLa细胞过程中发挥重要作用,ompA基因敲除菌株的获得为进一步研究其功能提供了帮助。     

应用酶联免疫吸附试验检测肠出血性大肠杆菌O157:H7

目的制备和纯化O157：H7抗原，免疫家兔和豚鼠，获得高效价的抗O157：H7免疫血清并进行纯化及酶标记。建立对O157：H7感染患者快速诊断方法，做到早期发现及时治疗，有效控制疫情的蔓延。方法ELISA双抗体夹心法检测O157：H7抗原。步骤：包被特异性抗体，加处理后的粪便等标本，然后加入抗O157：H7酶结合物，最后加入底物显色。结果本课题所研制的抗O157：H7酶结合物只对O157：H7呈阳性反应，而与其他相关细菌无交叉反应。结论应用酶联免疫吸附试验(即双抗体夹心法)对肠出血性大肠杆菌O157：H7抗原的检测较常规法实验程序简捷、快速、敏感。临床和现场验证结果表明，其方法具有灵敏度高，特异性强操作简单等特点，为肠出血性大肠杆菌O157：H7的鉴定及快速诊断提供了一种新的检测手段。     

尿道致病性大肠杆菌新基因R049特征的研究

目的 明确从国内分离的尿道致病性大肠杆菌(UPEC) 132中发现的新基因R049的特征及其表达蛋白在菌体内的定位。方法 提取UPEC132染色体DNA,鸟枪法构建文库,高通量焦磷酸法测序,Newbler软件拼接,进行生物信息学分析,确定R049相关片段的特征。提取UPEC132的内膜和外膜蛋白,与其全菌裂解物一起进行SDS-PAGE,用R049重组蛋白抗血清进行Western blot,确定R049表达蛋白的菌体内位置。结果 成功构建UPEC 132染色体文库,获取R049-contig169022 bp,与UPEC536染色体第233074至451502位碱基序列同源性较高,其中含有R049基因的20773 bp片段替代相当于UPEC536染色体上PAIⅢ536的位置,G+C含量为46.97％,两端具有正向重复序列,并与插入元件整合酶基因和thrW tRNA基因相邻,包含25个ORF,命名为R049 -GI。R049基因位于R049-GI的第13个ORF。SDS-PAGE和Western blot分析显示R049基因表达相对分子质量为47.0× 103蛋白是UPEC132的外膜蛋白。结论 R049基因编码细菌的外膜蛋白,是UPEC132通过基因水平转移获得的染色体基因组岛的组成部分。     

酶联免疫技术在乙型肝炎表面抗原检测中的应用

目的 分析酶联免疫吸附法（ELISA）在乙型肝炎表面抗原（HBsAg）检测中的临床应用价值.方法 选择150例乙肝患者和50例健康体检者,分别采用ELISA法、胶体金试纸条法、电时间分辨免疫荧光法（TRFIA）检查血液样本HBsAg,比较三种检查方法检查结果的一致性.结果 三种方法在检测乙型肝炎血清标志物HBsAg结果方面具有很高的一致性.结论 酶联免疫吸附法（ELISA）是检测乙型肝炎表面抗原（HBsAg）的有效方法,具有较高的临床应用价值,可以用于检验科的常规检查.     

布鲁氏菌外膜蛋白免疫蛋白质组学方法的建立

目的 建立布鲁氏菌外膜蛋白的免疫蛋白质组学研究方法,筛选布鲁氏菌保护性抗原.方法 利用双向电泳技术对实验条件下培养的布鲁氏菌疫苗株M5外膜蛋白进行分离,结合WB(Western blotting)技术寻找发生免疫反应的蛋白质.结果 21个免疫蛋白点经胶内酶切、肽提取后用基质辅助激光解析/电子飞行时间质谱(MALDI-TOF-MS)进行鉴定.每个蛋白点的肽质量指纹图谱用Mascot进行检索后,代表了12个开放阅读框.这些蛋白不全是外膜蛋白,还存在胞浆蛋白,其功能涉及生物合成和物质代谢等领域,还有一个功能未知的蛋白.结论 成功建立了布鲁氏菌外膜蛋白的免疫蛋白质组学研究方法,为寻找保护性抗原及为新型疫苗抗原候选提供新思路.     

Passive immunization with antibodies against iron-regulated outer membrane proteins protects turkeys from Escherichia coli septicemia.

Escherichia coli septicemia is a common disease of young poultry and several species of mammals. Rabbit antiserum was prepared against iron-regulated outer membrane proteins of E. coli. Eighteen-day-old turkeys were passively immunized with antiserum and challenged by air sac inoculation of 1 X 10(6) to 2 X 10(6) CFU of E. coli O78:K80:H9. Turkeys injected with normal rabbit serum or saline solution before challenge served as controls. Fatalities (8 of 51 turkeys inoculated) occurred only in groups given saline solution or normal rabbit serum. The remaining turkeys were necropsied 96 h after challenge. Passive immunization with antiserum significantly (P less than 0.05) reduced the frequency of bacteremia at 96 h after challenge, the frequency of recovery of E. coli from air sacs, and the severity of gross lesions in inoculated birds as compared with birds given normal rabbit serum or saline solution.     

Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.     

Antibodies raised against the outer membrane protein interrupt adherence of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAggEC) is a distinct category of diarrheal pathogen implicated as the cause of persistent diarrhea. The pathogen exhibits a characteristic "stacked-brick" pattern of aggregation when incubated with HEp-2 cells. The outer membrane protein (OMP) profile of a prototype EAggEC strain (F03) reflected the presence of one major 30-kDa protein. The OMP is expressed in the presence of the 60-MDa plasmid that the strain harbors. Antibodies were raised against the OMP by injecting the protein into a rabbit. The manifestation of an adherence phenotype on HEp-2 cells was observed for F03 and other strains that express OMP in the presence and absence of anti-OMP serum. Clumps of bacteria forming an aggregative pattern were observed in the HEp-2 cell assay in the absence of OMP antibodies, whereas a few bacteria attached to the cells in the presence of OMP antibodies. Mannose-resistant hemagglutination of human erythrocytes observed in the presence of EAggEC strains was inhibited in the presence of anti-OMP serum. Sequence analysis of a peptide generated by protease digestion of OMP exhibited 90% homology to a peptide of flagellin protein encoded by the hag gene of Serratia marcescens. Immunolabeling of the outer membrane by colloidal gold confirmed the protein to be an OMP. Our results suggest that the OMP of EAggEC have common antigenic properties. Antibodies raised against the protein can prevent adherence in vitro and could potentially interrupt the natural disease.     

Flagellin-deficient outer membrane vesicles as adjuvant induce cross-protection of Salmonella Typhimurium outer membrane proteins against infection by heterologous Salmonella serotypes

Salmonella enteric serovar infections result in high morbidity and mortality worldwide. Cross-protective vaccines are an effective strategy in controlling salmonellosis caused by multiple serotypes. In our previous study, outer membrane vesicles (OMVs) derived from flagellin-deficient Salmonella Typhimurium (S. Typhimurium) were proven effective in mediating cross-protection against infection by multiple Salmonella serotypes; OMVs also exhibit potent adjuvant effects. In this study, we further investigated the adjuvant capacities of flagellin-deficient S. Typhimurium OMVs. Our finding showed that outer membrane proteins (OMPs) in combination with flagellin-deficient S. Typhimurium OMVs could function as adjuvants and invoke stronger humoral, cellular, mucosal, and cross-protective immune responses compared to conventional aluminum (alum). Furthermore, as an adjuvant, OMVs could induce significantly higher cellular immune responses and display enhanced cross-protection for OMPs against wild-type virulent Salmonella Choleraesuis and Salmonella Enteritidis challenge. In summary, OMVs function as a potent adjuvant with the capability of conferring greater cross-protection against infection by multiple Salmonella serotypes, and may be of great value as an effective vaccine adjuvant in enteric diseases.     

Protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of Shigella spp.

Active immunization of guinea pigs and rabbits with outer membrane proteins (OMP) isolated from Shigella flexneri 3a and Shigella sonnei phase I protected the animals against keratoconjunctivitis shigellosa induced with the homologous or heterologous strain. Protection was also achieved in rabbits after passive immunization with anti-OMP immune serum. Active immunization with lipopolysaccharide of S. flexneri 3a did not protect rabbits against keratoconjunctivitis shigellosa.     

Serum antibodies to outer membrane proteins of Escherichia coli in healthy persons and patients with bacteremia

Antibodies to Escherichia coli outer membrane proteins in sera from healthy persons and from patients bacteremic with various enteric or nonenteric bacteria were measured by an enzyme-linked immunosorbent assay (ELISA). Outer membranes were prepared from E. coli O55. Serum was absorbed with E. coli O55 lipopolysaccharide and diluted 1:100 for immunoglobulin A (IgA) or IgM and 1:1,000 for IgG antibodies. Paired serum specimens were obtained from the 56 patients included in the study (the first specimen on the day of positive blood culture and the second specimen 8 to 12 days later) and compared with sera from blood donors (n = 50) as controls. On an average, the patients bacteremic with enterobacteria (n = 40) showed increased levels of antibodies of all three immunoglobulin classes in the first serum specimens and significantly higher levels in the second specimens compared with the controls, although with considerable case-to-case variation. Increased levels of IgG antibodies showed the best combination of diagnostic specificity (100%) and sensitivity (53%) for bacteremia caused by enteric bacilli. Mostly, the antibody response was directed against the major E. coli O55 outer membrane proteins at 81,000, 38,500, 33,500, and 7,500 molecular weights as shown by Western blot (immunoblot) analysis. Some of the patients bacteremic with nonenteric bacteria showed increased levels of IgA antibodies, but not of IgG or IgM antibodies. Cross-reactivity of the nonenteric blood culture isolates with the E. coli outer membrane preparation was not demonstrated. The cross-reactivity of the E. coli O55 outer membrane proteins with those of enteric bacilli of other genera was examined by absorption experiments. Western blots with serum absorbed with live E. coli O55 provided evidence that the epitopes of the outer membrane protein at 7,500 molecular weight were available for antibody binding at the bacterial surface, and that at least some of the epitopes of the 38,500- and 33,500-molecular -weight proteins were accessible to antibodies. The results suggest that an ELISA for the measurement of antibodies against cross-reactive outer membrane proteins from enteric bacilli may be useful in the diagnosis of serious infections caused by members of the family Enterobacteriaceae, and that antibodies to the major outer membrane proteins may have an immunobiological function.     

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.     

Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coli

Sera from normal healthy human adults and infants, as well as sera from mice, rabbits, and guinea pigs, were examined by immunoblotting for naturally occurring antibodies reacting with outer membrane proteins of two Escherichia coli strains, O111 and O18. Some individuals had antibodies reacting very strongly with the iron-regulated outer membrane proteins, including the ferric-enterochelin receptor protein (Mr, 81,000), as well as with ompA. However, sera from infants contained predominantly antibodies to ompA; antibodies recognizing the iron-regulated outer membrane proteins were either absent or barely detectable. In human serum the antibodies were mainly of the immunoglobulin G class. No serotype-specific antibodies to the lipopolysaccharide of E. coli O111 or O18 were found in the sera tested.     

Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa.     

Experimental infection of infant rabbits with verotoxin-producing Escherichia coli.

To study the pathogenesis of diarrheal disease due to verotoxin (VT)-producing Escherichia coli, 3-day-old rabbits were inoculated intragastrically with live E. coli O157:H7 (high VT producer), E. coli O113:K75:H21 (low VT producer), or O157:H45 (VT negative) and were examined for clinical symptoms, bacterial colonization, presence of detectable free VT in the intestines, and histological changes. Diarrhea developed consistently with 10(8) bacteria of E. coli O157:H7 but was observed only infrequently with even a higher dose of E. coli O113:K75:H21. VT-negative strains failed to cause diarrhea under the same experimental conditions. E. coli O157:H7 was recovered from the colon of infected animals in a significantly higher concentration than from the small intestine, and the clinical symptoms correlated with the presence of detectable free VT in the colon. Histological changes were seen mainly in the mid- and distal colon; these changes were characterized by a vast increase in apoptosis in the surface epithelium, increased mitotic activity in the crypts, mucin depletion, and a mild to moderate infiltration of neutrophils in the lamina propria and epithelium. Multiple foci of attached bacteria were seen on the surface epithelium of the gut-associated lymphoid tissue, cecum, and colon. Bacteria were never seen in epithelial cells or the lamina propria. These mucosal abnormalities as well as clinical symptoms were reproduced in infant rabbits by the intragastric administration of VT alone. These results are consistent with the hypothesis that VT plays a major role in the pathogenesis of diarrhea caused by E. coli O157:H7 and other VT-producing E. coli.