我国17株绵羊肺炎支原体分子分型及其菌体蛋白免疫印迹

【目的】分别从基因和蛋白水平研究我国部分地区绵羊肺炎支原体(Mycoplasma ovipneumoniae)分离株的分子特征,并了解其免疫原性蛋白的差异。【方法】对分离自8个地区的17株绵羊肺炎支原体进行扩增片段长度多态性(Amplified Fragment Length Polymorphism,AFLP)和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis,SDS-PAGE)分析,采用NTsys-2.10e软件对AFLP和SDS-PAGE结果进行聚类,并用绵羊肺炎支原体模式株Y98高免血清对部分分离株进行免疫印迹分析。【结果】当相似系数分别为0.78和0.85时,绵羊肺炎支原体分离株可根据8个来源地区分成8个AFLP群和8个SDS-PAGE群;用8株分离株进行免疫印迹共出现6条蛋白条带,分子质量分别为105 kDa、83 kDa、65 kDa、42 kDa、40 kDa或26 kDa,其中83 kDa和40 kDa蛋白为8个菌株保守的免疫原性蛋白。【结论】我国部分地区绵羊肺炎支原体分离株之间存在遗传差异,不同分离株的主要免疫原性蛋白也存在一定差异,但83 kDa和40 kDa蛋白为其保守的免疫原性蛋白。本研究首次对我国部分地区绵羊肺炎支原体分离株进行了分子分型与免疫印迹分析,结果将为绵羊肺炎支原体病的新型诊断技术开发和疫苗研制奠定理论基础。     

尼古丁在诱导的肺泡巨噬细胞自噬及肺炎中的作用

目的:探讨尼古丁在诱导的小鼠肺泡巨噬细胞自噬及肺炎发生中的作用。方法:通过碘化丙啶(PI)/Hochest33258染色法检测尼古丁诱导巨噬细胞MH-S死亡;通过Western印迹和电子透射显微镜检测尼古丁诱导MH-S细胞自噬的发生;采用中性红摄取实验检测尼古丁处理后巨噬细胞的吞噬能力;通过攻毒实验检测尼古丁诱导的肺炎。结果:不同浓度尼古丁作用下PI染色的死细胞呈上升趋势;LC3BⅡ蛋白表达具有尼古丁剂量依赖性,并且1μmol/L的尼古丁能够增强LPS预刺激的MH-S细胞自噬发生。电子透射显微镜结果显示在1μmol/L尼古丁的作用下,细胞内自噬体数量增加;MH-S的中性红摄取能力与尼古丁浓度剂量呈负相关性;尼古丁能够诱导小鼠发生肺炎,并降低小鼠体重。结论:尼古丁能够增强肺泡巨噬细胞的自噬水平,并且可以诱导肺炎的发生,有助于进一步研究吸烟与肺炎的关系。     

盘羊肺炎支原体感染PCR 检测方法的建立与初步应用

羊支原体性肺炎,是由支原体引起的一类高度接触性传染病,又称羊传染性胸膜肺炎(Infections pleuropneumonia of sheep and goats),临床症状主要表现为高热、咳嗽、消瘦、     

肺炎支原体感染的研究进展

肺炎支原体是引起呼吸道感染的常见病原体之一。近年来,肺炎支原体感染的发病率呈逐年增长趋势。肺炎支原体是介于病毒和细菌之间的原核生物,传播途径是呼吸道飞沫或气溶胶。感染该疾病后主要表现为上呼吸道感染、鼻咽炎、支气管炎、肺炎及严重的肺外并发症,如免疫性溶血性贫血、脑膜脑炎、心肌炎、心包炎、肾炎,严重感染者甚至可导致死亡。肺炎支原体有很强的传染性,经常在儿童集居地及家庭成员中交叉感染,导致久治不愈。对支原体肺炎进行早期诊断不仅可以避免并发症的发生率,也可遏制其继续传播。本文就肺炎支原体感染的致病机制及检测方法的研究,探讨了其对于支原体肺炎的早期诊断和病程监测的意义,将研究现状及展望作一综述。     

支原体巨噬细胞活化脂肽-2的研究进展

支原体巨噬细胞活化脂肽-2(MALP-2)是发酵支原体细胞膜上的一种脂多肽,能特异性识别靶细胞Toll样受体,产生免疫活性。MALP-2除了对机体和细胞有固有的促炎作用以外,还又具有对炎症的负调控作用,对机体的免疫系统也有一定的调节作用。就MALP-2的结构特征及其在疾病的发生、发展、治疗和预防等相关免疫学研究进展做一综述。     

猪肺炎支原体感染诱导的固有免疫应答研究进展

猪肺炎支原体是引起猪支原体肺炎的病原。由于缺乏成熟的猪肺炎支原体感染动物模型,使得猪肺炎支原体相关的抗感染免疫研究进展较为缓慢。本文从猪肺炎支原体感染后的炎症反应、固有免疫系统对猪肺炎支原体的识别、固有免疫细胞的作用、补体系统、抗菌肽、自噬以及细胞凋亡7个方面进行综述,旨在阐明固有免疫系统各组分在猪肺炎支原体感染中发挥的作用的研究进展,并对今后猪肺炎支原体感染的固有免疫应答研究的重点方向进行展望。     

儿童呼吸道肺炎支原体感染的实验室诊断

目的同时用目前常用的几种诊断肺炎支原体（MP）感染的实验方法检测MP,相互比较,得出适于常规诊断MP感染的方法或组合。方法搜集我院于2006年10月至2007年5月间以呼吸道感染入院儿童病例75例,采集其咽拭子和双份血清标本,以多种方法检测有无MP感染：培养法、EIA法测抗原、PCR法测MP-DNA,ELISA法测MP特异性IgG、IgA型抗体以及捕获法ELISA测MP特异性IgM型抗体。结果上述75例儿童中,共计有12例感染MP。以此为基础,上述各方法的敏感度分别是：培养法（25％）、EIA法测抗原（8．3％）、PCR法测MP-DNA（75．0％）、ELISA法测MP特异性IgA型抗体（单份血清为0,双份血清为33．3％）、捕获法ELISA测MP特异性IgM型抗体（单份血清为66．7％,双份血清为100％）。特异度分别是：100％、96．8％、93．7％（93．7％）、98．4％（98．4％）。PCR法和捕获法ELISA测MP特异性IgM型抗体结合后敏感度和特异度分别达到100％和95．2％。结论PCR法测MP-DNA和捕获法ELISA测MP特异性IgM型抗体的组合可高效地诊断MP感染,因而可作为临床诊断MP感染的一个常规组合。     

儿童哮喘与肺炎支原体感染关系的探讨

目的:探讨儿童哮喘发作与肺炎支原体(MP)感染之间的关系,并分析合并MP感染的患儿的临床表现。方法:将79例2-14岁急性哮喘发作的患儿依据病史分做两组:第一次哮喘发作的35人(始发哮喘组),已经有哮喘病史的44人(复发哮喘组)。采用被动冷凝集法检测两组患儿肺炎支原体抗体(MP-IgM)。结果:始发哮喘组和复发哮喘组分别有16例(45.7%)和10例(22.7%)患儿MP-IgM阳性(P0.05)。始发哮喘组与复发哮喘组MP-IgM阳性的患儿发热和肺部啰音发生率明显高于MP-IgM阴性的患儿(P0.05),血清IgE水平也明显高于MP-IgM阴性的患儿(P0.05)。结论:MP感染与儿童哮喘发作关系密切,合并MP感染的哮喘患儿发热或肺部啰音发生率明显高于未合并MP感染的哮喘患儿。     

肺炎支原体感染与咳嗽变异性哮喘的关系探讨

近来,每年都有肺炎支原体感染的散发和不同程度的流行。肺炎支原体(MP)已成为小儿呼吸道感染的重要病原。目前,肺炎支原体与咳嗽变异性哮喘(CVA)的关系越来越受到重视。本文通过对86例咳嗽变异性哮喘进行回顾性分析,现报告如下。1对象与方法1.1对象CVA组:2004年2月至2006年4月在台州市中心医院儿科门诊及随访的病例。所有病例均符合儿童咳嗽变异性哮喘防治常规的诊断标准[1],并血清支原体抗体检测治疗组45 0.61±0.22 6.2±1.5对照组43 0.83±0.38 9.1±1.3P值<0.05<0.05(日本富士株式会社生产,应用颗粒凝集法测定MP-IgM,MP-IgM≥1∶…     

绵羊肺炎支原体DnaK B细胞抗原表位预测及其蛋白结构分析

以绵羊肺炎支原体(Mo)热休克蛋白Hsp70(DnaK蛋白)氨基酸序列为基础,运用DNAStar软件和在线服务器等生物信息学分析工具,在同源性分析的基础上,通过Jameson-Wolf法、Kyte-Doolittle法、Emini法、Karplus-Schulz法及Welling法分别预测其抗原指数、亲水性、柔韧性、表面可极性等参数,综合分析、预测了该蛋白的B细胞抗原表位,并进行了蛋白的二级结构预测和3D结构模拟。结果表明,Mo贵州分离株的Hsp70蛋白与其它可致羊发病支原体的Hsp70蛋白同源性较低,说明该蛋白具有良好的特异性;Mo Hsp70蛋白整体抗原性较好,同时呈现较规则的空间结构,其中以C末端较稳定,区域也最大(第394-598区段),为优势B细胞抗原表位区域。     

Synthesis and immunomodulating activity of new glycopeptides of glycyrrhizic acid containing residues of L-glutamic acid

New glycopeptides of glycyrrhizic acid (GA) containing Glu residues and their α-methyl esters, γ-methyl esters, and α,γ-dimethyl esters were synthesized using N,N′-dicyclohexylcarbodiimide in the presence of N-hydroxybenzotriazole or N-hydroxysuccinimide. Formation of amide bonds was observed on all the three COOH groups of GA, or selectively on the COOH groups of the GA carbohydrate part in dependence on the ratio of reagents and the reaction conditions. The GA glycopeptide with three residues of Glu(OH)-OMe at a dose of 2 mg/kg stimulated the production of antibody-forming cells in mouse spleen in comparison with the control. The GA glycopeptide containing Glu residues only in the GA carbohydrate part turned out to be an immunosuppressor. The glycopeptide of the 30-methyl ester of GA with residues of free Glu in its carbohydrate part increased the hemagglutinine titer at oral doses of 2 and 10 mg/kg. All the studied compounds had practically no effect on the delayed-type hypersensitivity in mice.     

335弱碱性阴离子交换树脂对甘草酸的吸附过程

研究了335弱碱性阴离子交换树脂对甘草酸的吸附过程。拟合得到的吸附等温线方程为:c1/[q×(329-c1)]=0.035 8 1.872(c1/329),符合BET方程,计算得出335树脂的饱和吸附量是524.2 mg.g-1。通过吸附动力学曲线的研究,表明该树脂属于慢型吸附类型,得到树脂对甘草酸的吸附穿透曲线,穿透容量为42.00 mg.g-1,饱和容量近似为203.0 mg.g-1,交换柱的利用率小于0.206 9。用碱性洗脱液不易将树脂上吸附的甘草酸洗脱下来,利于甘草浸膏溶液中甘草酸和其它组分的分离。     

稀土元素对甘草细胞生长及甘草酸合成的影响

以胀果甘草根愈伤组织为材料,研究了在150mL摇瓶中,不同浓度的两种稀土离子镧、亚铈对甘草细胞生长及甘草酸分泌和释放的影响。结果表明,在悬浮培养初期、指数期加入稀土元素,改变了细胞生长状况,均使细胞在整体上生物量减少;不同种类的稀土离子、不同的稀土浓度对细胞生长影响强弱不同,但趋势相似。其中,培养指数期,加入500μL镧溶液的培养液中,所得甘草细胞生物量较多,利用薄层-紫外分光光度计法检测到甘草酸含量为67.68mg/g。     

Restoration of a bighorn sheep population impeded by Mycoplasma ovipneumoniae exposure

Bighorn sheep (Ovis canadensis) were once extirpated from the Black Hills region of South Dakota, U.S.A., mirroring declining populations throughout North America. Since the 1960s, several reintroductions have occurred in the Black Hills to reestablish populations, with varying success. We translocated 26 bighorn sheep from Alberta, Canada, to the Black Hills (February 2015) to restore bighorn sheep to their historic range. Due to prior examinations of cause‐specific survival, subsequent genetic diversity and disease prevalence analyses were required to evaluate success of the restoration effort. We measured a mean allelic diversity of 5.23 (SE = 0.44 [mean number of alleles]) and an observed heterozygosity of 0.71 (SE = 0.06; expected = 0.64 ± 0.05) in the translocated individuals. Translocated bighorn sheep tested negative for Mycoplasma ovipneumoniae at capture. An autogenous vaccine was administered prior to release in an attempt to safeguard the translocated bighorn sheep from infection with a strain known to be resident in adjacent bighorn sheep populations. However, the year following the translocation, a different strain of M. ovipneumoniae was associated with a pneumonia outbreak that resulted in 57.9% mortality. Our results suggest that allelic diversity and heterozygosity were sufficient for long‐term herd establishment, reducing the potential for founder effects. However, the overwhelming mortality associated with pneumonia, via the transfer of M. ovipneumoniae from an unknown source, limited the success or our reintroduction efforts. Successful attempts to restore bighorn sheep to their historic ranges must consider and mitigate potential routes for M. ovipneumoniae transmission pre‐ and post‐reintroduction.     

Development of selection method for Glycyrrhiza uralensis superior clones with high-glycyrrhizic acid contents using DNA sequence polymorphisms in glycyrrhizic acid biosynthetic genes

Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.     

Synthesis and biological activity of new glycyrrhizic acid conjugates with amino acids and dipeptides

New glycyrrhizic acid (GA) conjugates were synthesized with the use of tert-butyl esters of amino acids or benzyl esters of dipeptides; they contained two residues of L-amino acids (Met, Phe, Pro, and Ile or dipeptides Gly-Leu and Gly-Phe). Activation of GA carboxy groups was carried out with the help of N-hydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, or N-hydroxybenzotriazole with dicyclohexylcarbodiimide. A proline-containing GA derivative is a low-toxic substance; it raises the level of agglutinins by 3.7 times in the blood of mice and 3 times that of hemolysins compared with the control. Dipeptide GA derivatives possess an expressed anti-HIV-1 activity in cultures of MT-4 cells and are 90-70 times less cytotoxic than azidothymidine. The selectivity index of the compounds exceeds those of GA by 110 and 34 times, respectively.     

Simultaneous extraction and separation of liquiritin,glycyrrhizic acid,and glabridin from licorice root with analytical and preparative chromatography

Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice were developed by liquidliquid extraction with liquid chromatography separation. By utilizing different extraction solvents, procedures, and times, the optimum extraction conditions were established. The extracts of licorice were separated and determined using a C₁₈ column with a mobile phase consisting of acetonitrile-water (containing 1.0% acetic acid) with a gradient elution of 0∼10 min from 20:80 to 60:40 (v/v). Preparative columns with different packing sizes were investigated to isolate the three compounds from the extracts of licorice. The 12 μm chromatographic column showed better separation for the three compounds from licorice. 0.29 mg/g for liquiritin, 1.43 mg/g for glycyrrhizic acid, and 0.07 mg/g for glabridin were obtained and the recoveries were 80.8, 89.7, and 72.5%, respectively.     

Analysis of glycyrrhizic acid and liquiritin in liquorice root with microwave-assisted micellar extraction and pre-concentration

The feasibility of employing a non-ionic surfactant (Triton X-100) as an alternative and effective solvent for the microwave-assisted extraction of glycyrrhizic acid and liquiritin from liquorice root has been demonstrated. When compared with commonly used solvents, 5% Triton X-100 yielded higher extraction efficiency than aqueous solutions of ethanol or methanol. Under optimal conditions, i.e. 5% Triton X-100 (v/v) and microwave-assisted extraction for 3-5 min at 100 degrees C, the percentage extraction of active ingredients reached the highest value. The pre-concentration factor for the glycyrrhizic acid and liquiritin was about 13, and the cloud-point extraction recoveries for the two ingredients were 98.4 and 96.1%, respectively. The results showed that the coupling of microwave-assisted extraction and cloud-point extraction could be employed as a new and effective approach for the rapid extraction and pre-concentration of pharmacologically active ingredients from liquorice root without disturbing the subsequent chromatographic analysis.