血液杆菌Haematobacter sp.细胞脂肪酸测定的影响因素

应用美国MIDI公司Sherlock MIS系统检测一株归属于血液杆菌属的新菌种Haematobacter sp.HNMC11807的细胞脂肪酸组成,探讨不同培养方法和条件对其细胞脂肪酸组成的影响。采用不同培养条件培养微生物细胞,即不同的培养基组成、固液类型和培养时间;分析比较细胞脂肪酸检测结果存在的差异。同时对比分析仪器专用的两种检测体系,即常规标准和快速标准体系,对样品脂肪酸测定结果的影响。结果发现,细胞脂肪酸组成与其培养条件密切相关,且差异显著。因此,在应用该系统做微生物分类鉴定时,必需严格遵守特定的、统一的培养条件平行进行。

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

ABH and Lewis antigen distributions in blood,saliva and gastric mucosa and H pylori infection in gastric ulcer patients

AIM： TO investigate the ABH and Lewis antigen expression in erythrocytes, saliva and gastric epithelium, as well as the association between H pylori and the presence of gastric epithelial lesions.METHODS： The distribution of ABH and Lewis blood group antigens in erythrocytes, saliva and gastric mucosa of Hpylori-infected gastric ulcer patients was analyzed. Forty-two patients with gastric ulcer were studied, and fifty healthy individuals were used as control group. The blood group antigens were determined by direct hemagglutination, dot-ELISA and immunohistochemi- cal methods in erythrocytes, saliva and gastric mucosa specimens, respectively. Diagnosis for H pylori infection was performed by conventional optical microscopy and ELISA.RESULTS： A higher seroprevalence of IgG H pylori specific antibodies was observed in gastric ulcer patients （90%） compared to the control group （60%）. We observed a significant increase of phenotypes O, A2 and Lewis b in H pylori-infected patients. The expression of these antigens had progressive alterations in areas of ulcerous lesions and intestinal metaplasia.CONCLUSION： ABH and Lewis blood group antigens are a good indicator for cellular alterations in the gastric epithelium.

Validation of four Helicobacter pylori rapid blood tests in a multi-ethnic Asian population

AIM: To validate the accuracy of four rapid blood tests in the diagnosis of Helicobacter pylori.METHODS: Consecutive dyspeptic patients scheduled for endoscopy at the National University Hospital,Singapore, were interviewed and had blood drawn for serology. The first 109 patients were tested with BM-test (BM), Pyloriset Screen (PS) and QuickVue (QV), and the next 99 subjects were tested with PS and Unigold (UG).Endoscopies were performed blinded to rapid blood test results and biopsies were taken for culture and rapid urease test. Urea breath tests were performed after endoscopies. The rapid blood test results were compared with four reference tests (rapid urease test, culture,serology, and breath test).RESULTS: The study population composed of 208patients (mean age 43.1 years; range 18-73 years; 119males; 174 Chinese). The number of evaluable patientsfor BM, QV, UG and PS were 102, 102, 95, and 197,respectively. The sensitivity and specificity, respectively were: PS 80.2%, 95.8%; UG 55.9%, 100%; QV 43.3%,100%; BM 67.2%, 97.1%.CONCLUSION: The rapid blood test kits showed high specificity and positive predictive value (97-100%), while sensitivity and negative predictive value ranged widely (43%-80% and 47%-73%, respectively). Among test kits, PS showed the best sensitivity (80%), best negative predictive value (73%) and best negative likelihood ratio (0.207). PS had a specificity of 96%, positive predictive value of 97% and positive likelihood ratio of 19.1.

活动性肺结核病患者外周血ESAT6、Ag85B水平与细胞因子相关性分析

目的检测结核病患者外周血血清中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的水平,探讨分析ESAT-6、Ag85B水平与Treg细胞的增殖之间的相关性。方法 ELISA方法检测活动性结核病患者和健康志愿者外周血中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的表达水平,并用统计学方法分析血清中特异性分泌蛋白ESAT-6、Ag85B的浓度与Treg细胞刺激分泌的细胞因子IL-10、TGF-β1、IL-35的水平的相关性。结果活动性结核病患者血清中特异性分泌蛋白ESAT-6、Ag85B和细胞因子IL-10、TGF-β1的浓度与正常对照组相比均显著升高(P<0.001),但IL-35的浓度与正常对照组相比却显著降低,差异有统计学意义(P<0.001)。活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平与Treg细胞刺激活化分泌的细胞因子IL-10、TGF-β1、IL-35之间的相关性分析结果显示差异无统计学意义(P>0.05)。结论活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平均明显高于健康对照组,提示活动性结核病患者Treg细胞的高水平表达可能与结核分枝杆菌毒力系统有关。