肺炎链球菌假想蛋白SPD0873的表达纯化及保守性分析

目的通过构建原核表达载体,获得纯化的肺炎链球菌S．pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S．pn菌株中的保守性。方法分离培养D39型肺炎链球菌,获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32（a）内,测序鉴定。将重组质粒转化到E．coli Rossetta（DE3）中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni—NTA树脂纯化后,获得的重组蛋白用SDS—PAGE和Western印迹鉴定;将鉴定后纯化的蛋白免疫BALB／C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达。纯化蛋白免疫BALB／C小鼠获得高滴度、高特异性的的多克隆抗体,Western印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达。结论成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。     

肺炎链球菌触发肺Ⅱ型上皮细胞F-actin细胞骨架重排

目的：通过体外实验,研究肺炎链球菌（Streptococcus pneumoniae,S.pn)是否可触发肺Ⅱ型上皮细胞（A549）信号转导途径触发微丝肌动蛋白（filamentous actin,F-actin)细胞骨架重排,进而侵袭A549细胞,并初步分析触发A549细胞F-actin细胞骨架重排的细菌亚组分。方法：采用F-actin特异性FITC-phalloidin荧光染料,观察S.pn作用A549细胞前后的F-actin细胞骨架重排情况,依照重排百分率得分标准以（%）表示;用F-actin细胞骨架重排抑制剂细胞松弛素D预处理A549细胞,观察S.pn对A549细胞侵袭的改变情况;用变溶菌素提取S.pn细胞壁以观察其对F-actin细胞骨架重排的影响。结果：S.pn作用A549细胞后,经FITC-phalloidin荧光染色,F-actin细胞骨架呈黄绿色块状聚集,对照细胞呈现均匀黄绿色荧光外观;F-actin细胞骨架重排抑制剂细胞松弛素D可明显降低S.pn对A549细胞的侵袭,在其浓度为0.25μg/ml时,未得到可测的细菌数;S.pn细胞壁作用A549细胞后,经FITC-phalloidn荧光染色,F-actin细胞骨架呈黄绿色块状聚集,二者存在剂量依赖性。结论：S.pn及其细胞壁亚组分可触发A549细胞F-actin细胞骨架重排,进而侵袭A549细胞。     

肺炎链球菌溶血素黏膜免疫抗定植保护作用的研究

目的原核表达△A146Ply蛋白,评价△A146Ply黏膜免疫对肺炎链球菌（Streptococcuspneumon—ioe,5．Pn）在宿主鼻咽部定植的保护作用。方法IPTG诱导、Ni—NTA树脂纯化获得纯化的△A146Ply蛋白,经黏膜免疫BALB／C小鼠,制备其特异性抗血清;进行体内抗定植实验,观察小鼠鼻咽部灌洗液和肺部残存的细菌数量,检测△A146Ply黏膜免疫对19F型肺炎链球菌在鼻咽部定植的保护作用。为验证该保护作用是否具有广谱性,培养血清型14型、3型、6型和2型S．pn,经鼻腔感染免疫后小鼠,评价△A146Ply蛋白黏膜免疫对多株肺炎链球菌定植的保护作用。进行体外抗黏附实验,检测△A146Ply蛋白及其抗血清是否对无荚膜的肺炎链球菌R6黏附A549细胞具有抑制作用。结果获得了纯度〉90％的目的蛋白;体内实验结果显示,△A146Ply黏膜免疫可以显著降低肺炎链球菌19F在宿主鼻咽部和肺部残存的细菌数量（P〈0．01）;14型和3型肺炎链球菌在免疫后小鼠鼻咽部及肺部定植的数量均显著下降（P〈0．05）,2型肺炎链球菌在免疫后小鼠肺部定植的数量显著下降（P〈0．05）,6B型肺炎链球菌在免疫组与对照组小鼠鼻咽部及肺部均无显著差异（P〉0．05）;△A146Ply特异性抗血清△A146Ply蛋白对R6黏附A549细胞的抑制效应呈剂量依赖性。结论△A146Ply蛋白经黏膜免疫BALB／C小鼠可以对多种血清型的肺炎链球菌在宿主鼻咽部及肺部的定植提供显著保护作用,为Ply作为肺炎链球菌疫苗候选蛋白的应用提供了实验依据。     

肺炎链球菌触发人肺Ⅱ型上皮细胞F-actin细胞骨架重排与TPK信号转导的相关性研究

目的:通过体外实验,研究Streptococcus pneumoniae(S.pn)是否通过肺Ⅱ型上皮细胞(A549)酪氨酸蛋白激酶(TPK)信号转导途径触发微丝肌动蛋白(Filamentous actin,F-actin)细胞骨架重排,进而导致S.pn对A549细胞的侵袭.方法:采用F-actin特异性FITC-phalloidin荧光染料,观察S.pn作用A549细胞前后的F-actin细胞骨架重排情况,依照重排百分率得分标准以(%)表示;用F-actin细胞骨架重排抑制剂细胞松驰素D预处理A549细胞,观察S.pn对A549细胞的侵袭率;使用TPK信号转导抑制剂Genistein预处理A549细胞,观察其与F-actin细胞骨架重排百分率间是否存在剂量依赖关系.结果:S.pn作用A549细胞后,经FITC-phalloidin荧光染色,F-actin细胞骨架呈块状、丝状聚集;F-actin细胞骨架重排抑制剂细胞松驰素D可明显降低S.pn对A549细胞的侵袭率,在其浓度为0.25μg/ml时,未得到可测的细菌数;TPK信号转导途径抑制剂可部分抑制A549细胞F-actin细胞骨架重排,并与F-actin细胞骨架重排百分率间存在量效关系,其相关系数分别为rTpK=-0.91(P＜0.05).结论:上述结果提示S.pn可通过TPK细胞信号转导途径触发A549细胞F-actin细胞骨架重排,进而导致S.pn侵袭A549细胞.     

钙信号转导途径介导肺炎链球菌侵袭人肺Ⅱ型上皮细胞

用Factin 特异性FITCphalloidin荧光染料,观察肺炎链球菌（Streptococcus pneumoniae）作用A549细胞前后的Factin细胞骨架重排情况;用细胞松弛素D预处理A49细胞,观察肺炎链球菌对A549细胞的侵袭率;使用Datrolene预处理A549细胞,观察其与Factin细胞骨架重排百分率间是否存在剂量依赖关系;用Fura2/AM荧光探针负载A549细胞后测定肺炎链球菌粘附A549细胞后的胞内Ca2+浓度。结果发现肺炎链球菌作用A549细胞后,Factin细胞骨架呈块状、丝状聚集;而松弛素D可明显降低肺炎链球菌对A549细胞的侵袭率;肺炎链球菌粘附A549细胞后胞内Ca2+高于对照;Datrolene可部分抑制A549细胞Factin细胞骨架重排,且与Factin细胞骨架重排百分率间存在量效关系。以上结果提示肺炎链球菌可通过Ca2+细胞信号转导途径触发A549细胞Factin细胞骨架重排,进而导致肺炎链球菌侵袭A549细胞。     

通过替代失活的方法分析核酸片段在肺炎链球菌毒力中的作用

目的鉴定基因spd—ABC、spd-1672、sp-1673和长间隔序列spd—J在肺炎链球菌（Streptococcus pneumoniae,S．pn）毒力中的作用。方法采用长臂同源聚合酶链反应（LFH—PCR）的方法分别构建这4个序列的红霉素抗性基因（erm）替代缺失突变体,通过PCR和测序鉴定是否构建成功;通过绘制生长曲线观察基因对细菌生长繁殖的影响,通过小鼠毒力实验观察基因对细菌致病性的影响。结果所构建缺陷菌目的基因由eFm基因完全替代;spd—ABC、spd-1673和长间隔序列spd—J3个片段缺陷菌的生长趋势与野生菌没有明显差异,生长大约5hA值均可达到峰值,而spd-1672缺陷菌出现明显的延迟现象,生长8h后其4值才达到最高值;野生菌与缺失spd—ABC、spd-1673和长间隔序列spd—J的缺陷菌感染小鼠各组半数死亡时间分别为19、22、24和24h,差异无统计学意义,而spd-1672缺陷菌感染小鼠的半数死亡时间在75h左右,显著长于野生菌感染小鼠组（P〈0．05）。结论在构建的4个单个基因缺失突变体中,spd-1672缺陷后．S．pn生长明显延迟,毒力显著下降,提示spd-1672是s-Pn的一个新的毒力因子。     

一种溶菌酶样蛋白对肺炎链球菌毒力及荚膜多糖合成的影响

摘要：【目的】为了研究肺炎链球菌（Streptococcus pneumoniae, S.pn）的一种假想的溶菌酶样蛋白在细菌生物学性状及其致病中的作用。【方法】利用长臂同源PCR对该基因进行敲出,并同时构建带有拯救质粒的缺失菌株,观察D39野生菌、缺失菌与带有拯救质粒的缺失菌株在相关生物学性状及其致病力改变,从而鉴定这种假想溶菌酶样蛋白的功能。【结果】缺失菌与野生菌相比,细菌生长减缓,毒力下降,荚膜多糖合成明显减少。而将拯救质粒转入缺失菌株后,该溶菌酶样蛋白的mRNA表达水平较野生菌高,其毒力及荚膜合成     

C型凝集素LSECtin识别细菌的研究

目的:通过sLSECtin-Fc蛋白与细菌及细胞的黏附,寻找能与LSECtin结合的细菌。方法:通过ELISA检测sLSECtin-Fc与细菌的黏附,同时构建CHO-pDsRed1-N1-LSECtin稳定细胞株进行与细菌的黏附实验,荧光显微镜进行镜鉴拍照。结果:获得能够与sLSECtin-Fc蛋白黏附的细菌,并且通过过表达LSECtin的稳定株与细菌的黏附,发现LSECtin可以结合大肠杆菌和肺炎链球菌,而不能结合金黄色葡萄球菌和肺炎克雷伯杆菌。结论:发现LSECtin可以结合细菌,为进一步研究LSECtin在先天免疫中的作用奠定了基础。     

肺炎链球菌粘附机制的研究现状

肺炎链球菌粘附宿主我肺炎链球菌侵袭、感染宿主细胞的先决条件。粘附过程是特异的,是细菌表面的粘附分子和宿主细胞膜受体相互作用的结果。英膜对肺炎链球菌的粘附无影响,而细胞壁（ＣＷ）在介导肺炎链附粘附宿主细胞过程中起重要作用;ＣＷ亚组人脂磷酸（ＬＴＡ）介导肺炎链球菌的粘附过程,并导致炎症反应;细菌表面的结构蛋白或分泌蛋白是细菌与宿主细胞连接的桥梁;肺炎链球菌能与宿主细胞外基质蛋白特异性结合,进而粘附宿主     

一种基于绿色荧光蛋白的蛋白酶体抑制剂细胞筛选模型

为建立基于绿色荧光蛋白(GFP)的药物筛选模型,并用此模型从包括中药提取物在内的化合物中筛选新型蛋白酶体抑制剂,本研究构建了pGC-E1-ZU1-GFP融合蛋白慢病毒表达载体并感染A549细胞,筛选稳定表达细胞株,用已知蛋白酶体抑制剂PS-341处理细胞,荧光显微镜检测处理前后细胞GFP水平变化。结果获得了稳定表达pGC-E1-ZU1-GFP的A549细胞,这些细胞用PS-341处理24h后用荧光显微镜检测,发现细胞绿色荧光强度相对于对照组明显增强。利用这一模型对一些化合物进行筛查,发现了一些新的蛋白酶体抑制剂。     

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.     

Construction of Improved Tools for Protein Localization Studies in Streptococcus pneumoniae

We have constructed a set of plasmids that allow efficient expression of both N- and C-terminal fusions of proteins of interest to fluorescent proteins mCherry, Citrine, CFP and GFP in the Gram-positive pathogen Streptococcus pneumoniae. In order to improve expression of the fluorescent fusions to levels that allow their detection by fluorescence microscopy, we have introduced a 10 amino acid tag, named i-tag, at the N-terminal end of the fluorescent proteins. This caused increased expression due to improved translation efficiency and did not interfere with the protein localization in pneumococcal bacteria. Localizing fluorescent derivatives of FtsZ, Wzd and Wze in dividing bacteria validated the developed tools. The availability of the new plasmids described in this work should greatly facilitate studies of protein localization in an important clinical pathogen.     

不同强度高压电场对A549肺癌细胞肿瘤生物学特性的影响

目的：探讨不同强度高压电场对A549肺癌细胞肿瘤转移生物学特性的影响。方法：选择处于生长周期的A549细胞,共分为7个组进行研究,其中A-F组为实验组,G组为不施加电场的空白对照组,A施加500V/cm强度高压电场,F组施加1750V/cm的高压电场,电压场强间隔为250V/cm。采取粘附实验、侵袭及转移实验,检验A549细胞在不同强度高压电场中,其肿瘤生物学转移特性的改变。结果：①各实验组与对照组、各实验组之间的细胞粘附能力,均存在显著性差异（P〈0.05）;②电场强度≥750V/cm时,各实验组之间、及其与对照组之间的细胞迁移能力,存在显著性差异（P〈0.05）;③电场强度≥1000V/cm时,各组与对照组间的细胞侵袭能力,存在显著性差异（P〈0.05）;④电场强度为1000-1250V/cm的各组与1500-1750V/cm各组间的细胞侵袭能力,存在显著性差异,有统计学意义（P〈0.05）。结论：不同强度的电场抑制A549肺癌细胞的程度不同,随着强度的增加,A549细胞粘附、迁移和侵袭能力的抑制现象依次出现,并随着电场强度的增加其抑制程度也持续增加。     

绿色荧光裸鼠的建立和验证

目的建立系统性表达绿色荧光蛋白的裸鼠,接种人源肺癌细胞验证该模型是否具有免疫缺陷性,并观察双色荧光的成像效果。方法利用系统性表达绿色荧光蛋白的C57BL/6J小鼠与BALB/C裸小鼠多代杂交和互交,建立稳定表达绿色荧光蛋白的裸鼠。大体解剖观察胸腺生长情况,整体和器官荧光成像验证绿色荧光蛋白的表达情况。以2×106/只的剂量对其皮下腋下接种表达红色荧光蛋白的人类A549肺癌细胞（RFP-A549）,通过观测肿瘤生长来验证模型的免疫缺陷性。同时,利用红色荧光标记的肿瘤和绿色宿主鼠,对双色的整体成像效果进行观测。结果构建出系统性表达绿色荧光蛋白的裸鼠,大体解剖可见胸腺缺失。在激发光的激发下,绿色荧光裸鼠全身发出清晰的绿色荧光,脑、心脏、肺脏、肝脏、肾脏,肠胃及胰腺等主要器官可见明显绿色荧光。接种RFP-A549细胞后,成瘤率达到100%,整体动物荧光成像表现出清晰的双色。结论本研究构建出的绿色荧光裸鼠,动物整体可以清晰地表达绿色荧光并具有免疫缺陷性     

Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae

Opa protein-expressing pathogenic neisseriae interact with CD66a-transfected COS (African green monkey kidney) and CHO (Chinese hamster ovary) cells. CD66a (BGP) is a member of carcinoembryonic antigen (CEA, CD66) family. The interactions occur at the N-terminal domain of CD66a, a region that is highly conserved between members of the CEA subgroup of the CD66 family. In this study, we have investigated the roles of CD66 expressed on human epithelial cells and polymorphonuclear phagocytes (PMNs) in adhesion mediated via Opa proteins. Using human colonic (HT29) and lung (A549) epithelial cell lines known to express CD66 molecules, we show that these receptors are used by meningococci. A monoclonal antibody, YTH71.3, against the N-terminal domain of CD66, but not 3B10 directed against domains, A1/B1, inhibited meningococcal adhesion to host cells. When acapsulate bacteria expressing Opa proteins were used, large numbers of bacteria adhered to HT29 and A549 cells. In addition, both CD66a-transfected CHO cells and human epithelial cells were invaded by Opa-expressing meningococci, suggesting that epithelial cell invasion may occur via Opa–CD66 interactions. In previous studies we have shown that serogroup A strain C751 expresses three Opa proteins, all of which mediate non-opsonic interactions with neutrophils. We have examined the mechanisms of these interactions using antibodies and soluble chimeric receptors. The results indicate that the nature of their interactions with purified CD66a molecules and with CD66 on neutrophils is alike and that these interactions occur at the N-terminal domain of CD66. Thus, the Opa family of neisserial ligands may interact with several members of the CD66 family via their largely conserved N-terminal domains.     

Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.     

Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells

Benzylisoquinoline alkaloids are a large and structurally diverse group of natural plant products that includes many compounds with potent biological activities, including the antimicrobial agent sanguinarine. The putative subcellular localization of the sanguinarine pathway was determined using in-frame N-terminal fusions between cDNAs encoding nine consecutive biosynthetic enzymes and the gene encoding the green fluorescent protein (GFP). Expression constructs were introduced into cultured opium poppy cells by particle bombardment, and the localization of fusion proteins was visualized using epifluorescence microscopy. GFP fusions with two O-methyltransferases and two N-methyltransferases in the sanguinarine pathway all produced non-targeted fluorescence in the cytosol and nucleus. Interspersed between these soluble proteins are five membrane-bound cytochromes P450. Corresponding cDNAs are available for three P450s, all of which produced fluorescence when fused to GFP in association with the endoplasmic reticulum (ER). Two enzymes with suggested or known N-terminal signal peptides were initially associated with the ER, but were subsequently transported to the central vacuole suggesting their occurrence in the ER lumen. The alternating localization of these biosynthetic enzymes to three subcellular compartments indicates extensive trafficking of pathway intermediates across the endomembranes and suggests a key role for compartmentalization in the regulation of sanguinarine metabolism.