缓释破伤风类毒素微球的制备及其免疫原性

目的 用可生物降解缓释微球,单剂投递破伤风类毒毒。方法 用溶剂挥发法,将破伤风类毒素(TT)包入两种丙交酯-乙交酯共聚物制备成微球,用电镜观察微球的粒经与表面形态,用Bradford法及SDS-PAGE检测微球内蛋白含量及抗原结构的变化。TT微球免疫豚鼠后,观察抗体应答。结果 微球表面光滑,成球规律,两种微球平均粒径为8.4μm及9.7μm,包裹率为48％及56％,包裹后的TT结构未改变。TT微球免疫豚鼠后,1针诱导的抗TT-IgG及中和抗体滴度与3针铝吸附TT相似;TT微球免疫血清及铝吸附TT免疫血清与游离破伤风毒素有相似的结合特性。加强免疫后,TT微球的回忆应答优于铝佐剂疫苗。结论 可生物降解缓释微球单剂投递破伤风类毒素,可产生持续高的免疫应答。

IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达

目的 :IL - 2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达。方法 :分别在金黄色葡萄球菌肠毒素A2 2 7Ala、B基因的两端克隆上两个酶切位点HindⅢ ,KpnⅠ。将IL - 2基因突变 ,设计一段linker使之分别与SEA2 2 7Ala和SEB相连并克隆到PET表达载体中 ,在大肠杆菌DH5α(DE3) -Pass中表达。结果 :表达的蛋白占总蛋白 15 %。结论 :IL -

北京某区一起疑似金黄色葡萄球菌肠毒素中毒分析

目的 通过实验室检测及全基因组测序,分析一起金黄色葡萄球菌肠毒素(SEs)中毒,为应对此类食源性暴发事件提供经验。方法 采集可疑受污染食品、病例肛拭子和环境涂抹样本,采用金黄色葡萄球菌5种肠毒素(SEA、SEB、SEC、SED、SEE)的酶联免疫吸附实验(ELISA)检测及肠毒素基因(sea、seb、sec、sed、see)荧光PCR方法检测;对分离株进行耐药表型检测和全基因组测序并构建进化树。结果 3件可疑受污染的食品未检出SEs。荧光PCR检测3件可疑污染食品的增菌液结果都为nuc+/seb+。从3件可疑受污染食品中共分离到9株seb+金黄色葡萄球菌,其中从食品1中分离到2株t78型和1株t437型,食品2和食品3中分离到的菌株均为t437型,同一spa分型菌株具有相同耐药表型。从1件环境涂抹中分离出2株5种肠毒素基因均为阴性的金黄色葡萄球菌,均为t571型。由9株食品分离株和2株环境分离株构建cgSNP进化树,显示形成3个独立且遗传关系较远分支。9株食品分离株产毒培养后ELISA肠毒素检测结果为SEB阳性。结论 本起事件为可疑SEB中毒事件,全基因组测序可提供较为全面的生物信息数据以应对此类事件。

杀线虫真菌──粗皮侧耳初报

通过研究报道了一类高效杀线虫真菌，其分类地位为担子菌亚门、侧耳属（Pleurotus）．粗左侧耳（P．ostreatus）。该菌毒力强、见效快，数分钟内可将线虫击倒，其作用主要是真菌毒素。在WA，CMA和PDA平板上，菌丝体均能产生大量毒素球，加线虫可刺激毒素球大量增加，但菌体液体培养不产生毒素。致病性测定表明，该菌对腐生线虫和寄生线虫都具致病力。Panagrellus redi－vivus与毒素接触1～5min停止活动，最终线虫死亡，菌丝侵入虫体，1—3d消解线虫。菌丝体处理二令爪哇根结线虫（Miloidogyne javanica）10d，P．redivivus线虫4d，线虫死亡率均达99％，对照死亡率为0～4％。该菌菌种易于保存和提纯复壮。

Temperature-Induced Unfolding Pathway of Staphylococcal Enterotoxin B:Insights from Circular Dichroism and Molecular Dynamics Simulation

In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.

粗皮侧耳真菌能杀死线虫

华南农业大学向红琼和贵州大学张克勤对粗皮侧耳真菌杀线虫效果进行了研究。结果表明,高效杀线虫真菌的分类为担子菌亚门、侧耳属(Pleurotus)、粗皮侧耳(P.ostreatus)。该菌毒力强,见效快,数分钟内可将线虫击倒,其作用主要是真菌毒素。在WA、CMA和PDA平板培养基上,菌丝体均能产生大量毒素球,加线虫后可刺激毒素球大量增加,但菌体液体培养不产生毒素。致病性测定表明。

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Cell-specific expression of the diphtheria toxin A-chain coding sequence induces cancer cell suicide

OBJECTIVE: To test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells. METHODS: The diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed. RESULTS: The beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells.Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ. CONCLUSION: Selective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.