铜绿假单胞菌外毒素A的表达、纯化与生物学活性研究

目的利用基因工程技术制备铜绿假单胞菌外毒素A(PE),为深入研究PE的致病机理和免疫防治奠定基础。方法应用PCR技术从铜绿假单胞菌基因组中扩增外毒素A全长结构基因,将其克降于原核表达载体pQE-31中,所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109, IPTG诱导表达;制备融合蛋白包涵体,并采用Ni-NTA柱亲和层析、葡聚糖凝胶过滤和阴离子交换层析分离和纯化目的蛋白;采用透析法对纯化后的目的蛋白进行复性,MTT法测定复性后的重组PE对L929细胞、B16黑素瘤细胞的细胞毒活性。结果通过对PCR反应体系的优化,扩增到了PE全长结构基因。所构建的pQE-PE重组质粒经酶切及测序鉴定与设计序列一致;转化E.coli JM109后,IPTG诱导目的蛋白表达率约为25%;SDS-PAGE初步测定目的蛋白的相对分子质量(M_r)约为66×10~3,与理论预期值一致;破菌后电泳证实目的蛋白主要以包涵体形式表达。经亲和层析、葡聚糖凝胶过滤和阴离子交换层析后蛋白纯度大于95%。MTT法测得重组PE对L929细胞和B16黑素瘤细胞的半数抑制浓度(IC_(50)值)分别为2.13μg/ml、2.58μg/ml。结论通过对PE的表达纯化,获得了具有细胞毒活性的重组PE,为利用基因工程手段大量制备PE的工作奠定了基础。     

PA感染与定植中细胞因子变化的研究

目的探讨铜绿假单胞菌外毒素(PEA)在铜绿假单胞菌肺部感染及定植表达差异的临床价值。方法 SD大鼠180只,随机分为PA(铜绿假单胞菌)感染模型组,PA咽部定植组及生理盐水对照组。取肺组织HE染色光镜下观察肺部炎症情况。结果①细菌培养结果:模型组在接种后肺组织PA菌落数明显升高;定植组咽拭子培养见大量PA生长;定植组及对照组肺组织未见PA菌生长;②模型组血清IL-6、IL-8、IFN-γ、TNF-α表达在接种后第3、7、11天均明显高于定植组及对照组(其差异有统计学意义)。结论血清细胞因子IL-6、IL-8、IFN-γ、TNF-α检测有助于对铜绿假单胞菌感染与定植的鉴别诊断。     

抗绿脓杆菌外毒素—A血浆（马）抗体效价检测方法的探讨

目的：为了探讨在生产条件下采用哪种方法来检测抗绿脓杆菌外毒素—A血浆（马）的抗体效价更为可行。方法：本文对小鼠中和试验，双向琼脂扩散试验及胶乳凝集试验进行了比较研究。结果实验显示，小鼠中和试验和双向琼脂扩散试验特异性良好，结果稳定，但前者灵敏度比后者更高。而胶乳凝集试验由于胶乳抗原不易保存，其结果稳定性较差。结论：我们认为小鼠中和试验适用于大规模生产时采用，双向琼脂扩散可作为初步效价的检测。     

Mutational effects on protein folding stability and antigenicity: the case of streptococcal pyrogenic exotoxin A

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.     

Staphylococcal superantigens and toxins are detectable in the serum of adult burn patients

Bacterial infection in burn patients is still a devastating contributor to morbidity and mortality. Little is known regarding the presence of staphylococcal toxins in the burn-injured patient. The aim of this study was to characterize the prevalence of several of these toxins and their relationship to clinical metrics and mortality in burn patients. Levels of exotoxins staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B, toxic shock syndrome toxin 1 (TSST-1), and α-hemolysin were assayed from the serum of 207 adult burn patients aged 16–92 years. Clinical, demographic, and microbiological data from these patients were then compared to toxin levels. Staphylococcal exotoxins α-hemolysin and SEA were present in 45% and 25% of the population, respectively. Bacterial cultures concomitantly showed a high prevalence of Staphylococcus aureus in 48% of patients, of which 59% were methicillin resistant. Several metrics may be predictive of high toxin concentrations of α-hemolysin and TSST-1 and SEA including burn size, length of stay, and bacteremia. Mortality associations indicated that burn size, bacteremia, age, and the presence of α-hemolysin and SEA may be predictors of mortality. A high prevalence of staphylococcal toxin α-hemolysin and superantigens TSST-1 and SEA can be found in the circulation of the adult burn population. The presence of these toxins may contribute to the morbidity and mortality of the burn patient.     

Aspergillus niger pneumonia with fatal pulmonary oxalosis

Metabolic products of Aspergillus species may play a significant role in the pulmonary destructive process. We describe a patient who died of respiratory failure, in whom postmortem examination revealed aspergilloma and numerous calcium oxalate crystals around the aspergilloma, as well as extensive consolidation areas. An-87-year-old man with a history of pulmonary tuberculosis and asbestos exposure was admitted to our hospital with fever and hemosputum. Chest radiograph on admission showed several small cavities in the right upper lung fields, but did not indicate the presence of a fungus ball. The patient was treated with several antibiotics, but his symptoms, and findings for inflammatory indicators and findings on chest radiographs deteriorated, and he died of respiratory failure 45 days after admission. Postmortem examination of the thoracic cavities showed marked involvement with extensive adhesions, fibrosis, caseation, and necrotic tissue. Aspergillus niger formed a mass in the right upper cavity and a localized invasion surrounding the cavity wall, but no organisms were detected in the left consolidation area. Numerous calcium oxalate crystals were found in the cavity wall, as well as an extensive consolidation area. We consider that oxalic acid produced by Aspergillus was the main cause of the patient's respiratory failure. Received: July 23, 1998 / Accepted: December 21, 1998     

铜绿假单胞菌外毒素A全长基因的扩增与克隆

目的　从绿脓杆菌中提取染色体DNA ,PCR扩增及克隆外毒素A全长基因 ,为实现外毒素A的高效表达奠定基础。方法　从绿脓杆菌培养物中提取染色体DNA ,PCR扩增外毒素A全长基因 ,A -T克隆入本室自行构建的pSK -T载体中 ,对重组质粒进行酶切与测序鉴定。结果与结论　成功地从高GC含量的绿脓杆菌染色体DNA中扩增到长片段的外毒素A全基因并进行了克隆与鉴定。     

促黄体激素释放激素-绿脓杆菌外毒素A与胃癌细胞受体结合竞争的研究

目的探讨重组人促黄体激素释放激素-绿脓杆菌外毒素A(LHRH-PE40)在胃癌细胞及正常细胞是否存在相应受体表达及存在竞争性抑制。方法将胃癌细胞系BGC-823及正常人肾细胞系293制备成细胞膜,利用125I标记的LHRH-PE40与两种细胞膜进行放射性配基分析, 以及与LHRH竞争结合分析。结果人肾细胞系293未见配基-受体特异性结合;而人胃癌细胞系 BGC-823的结合竞争符合特异性配基-受体结合、竞争。BGC-823细胞亲和力:Kd=(37．82±1．42) nmol／L,容量Bmax=475．00±17．86 pmol／mg。结论 LHRH是胃癌免疫治疗的有效靶点,LHRH- PE40对过度表达LHRH受体的胃癌均具有特异有效的抗肿瘤活性。     

CM101, a polysaccharide antitumor agent,does not inhibit wound healing in murine models

CM101 (previously called GBS toxin), a new anticancer polysaccharide that induces inflammatory reactions in neovasculature of tumors, does not cause similar reactions in neovasculature of healing wounds. It appears that treatment with CM101 will not interfere with normal wound healing in cancer patients.Abbreviations PVA polyvinyl alcohol - PBS phosphate-buffered saline