申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建

目的 筛选与申克孢子丝菌酵母相与菌丝相双相转换相关的差异表达基因,为探讨其双相转换的分子的机制奠定基础.方法 应用抑制性消减杂交技术,构建高特异性的申克孢子丝菌菌丝相(mycelium,M)和酵母相(yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签...     

白念珠菌不同菌态成分诱导小鼠DCs分化成熟的差异性比较

白念珠菌是人类最常见的条件致病菌之一,可引起从皮肤黏膜到内脏的广泛感染.白念珠菌从形态观察为双相真菌,有酵母相和菌丝相,其中菌丝相更易黏附入侵宿主组织,是该菌在体内的主要致病形式.DCs是体内最重要的抗原提呈细胞,正常情况下绝大多数处于非成熟状态,一旦摄取抗原或接受某些因素刺激,DCs就会发育成熟,通过多种因素可诱导T细胞向不同类型Th细胞分化,形成抗感染免疫的起点和调节点.本研究通过将粉碎后白念珠菌酵母相、菌丝相成分分别刺激小鼠骨髓源DCs,观察DCs形态、表达表面分子CD80、CD86及分泌IL-12情况,比较两者间的差异.     

小鼠Dectin-1胞外区的融合表达及其识别白念珠菌β-葡聚糖的研究

目的 克隆、表达并纯化小鼠腹腔巨噬细胞Dectin-1基因胞外区,探讨其识别并结合真菌β-葡聚糖的能力.方法 应用RT-PCR方法从小鼠腹腔巨噬细胞RNA中扩增Dectin-1基因胞外区,构建原核重组表达载体pET28-CRD,进行融合表达、纯化和复性.将融合蛋白与白念珠菌酵母相共同孵育,检测其识别、结合白念珠菌细胞壁β-葡聚糖的功能.结果 成功克隆并构建原核重组表达质粒pET28-CRD,表达并纯化了融合蛋白,该蛋白能识别、结合白念珠菌酵母细胞壁β-葡聚糖.结论 构建的原核重组表达载体能够在原核细胞内表达,表达产物有识别、结合真菌胞壁β-葡聚糖的功能,为进一步研究相应的真菌检测方法奠定了基础.     

Tyrosol和Farnesol对白念珠菌生物被膜形成作用初探

目的 研究密度感应分子(quorum sensing molecule)tyrosol(对羟基苯乙醇)和farnesol(法呢醇)对白念珠菌生物被膜形成的调控作用.方法 在tyrosol和farnesol干预下构建白念珠菌临床株和标准株生物被膜,在倒置显微镜下观察细胞形态,应用RT-PCR技术检测密度感应分子对白念珠菌HTA1和EFG1基因表达的调控作用,并采用MTT法观察密度感应分子对细胞活性的影响.结果 tyrosol对白念珠菌生物被膜的菌丝发生和细胞活性无明显促进作用,也无法中和farnesol对菌丝发生和细胞活性的抑制作用.tyrosol使白念珠菌生物被膜内细胞HTA1的表达增强,对EFG1的表达并无明显影响;tyrosol不能改变famesol对HTA1和EFG1表达的抑制作用.结论 tyrosol能在一定程度恢复口腔白念珠菌生物被膜内细胞的活跃状态,但当tyrosol与famesol同时存在时,tyrosol的作用被后者的抑制效应所掩盖,细胞对farnesol更敏感.     

痢疾菌体内诱导基因表达文库的构建

目的 构建筛选痢疾菌福氏2a体内诱导基因的融合基因表达文库。方法 以自杀载体pGP704为骨架，用氯霉素乙酰转移酶基因作为报告基因，构建了用于筛选体内诱导基因的载体pGP-cat。把痢疾菌福氏2a基因组DNA的随机酶切片段（0．6－1kb)亚克隆到pGPcat载体报告基因上游的BglⅡ位点，构建了融合基因文库。结果 与痢疾菌福氏2a2457T结合转移后，获得融合基因表达文库。以氯霉素为选择标记，经过体外初步筛选，得到3．2％的具有氯霉素抗性的菌株。结论 构建的融合基因表达文库适用于筛选福氏2a痢疾菌的体内诱导基因。     

抑制性消减杂交技术筛选重组IFNβ下调HepG2细胞靶基因

目的利用抑制性消减杂交（SSH）技术构建重组干扰素β（IFNβ）刺激人肝癌细胞系HepG2差异表达基因的cDNA消减文库，筛选IFNβ下凋HepG2相关基因。方法重组IFNβ2000U／ml刺激对数生长期HepG2细胞，以生理盐水作用的HepG2细胞为阴性对照；制备细胞裂解液，从中提取mRNA并逆转录为cDNA，经Rsa Ⅰ酶切后将实验组cDNA分成两份，分别与两种不同的接头连接，再与对照组cDNA进行两次消减杂交及两次抑制性PCR，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠埃希菌进行文库扩增，随机挑选克隆PCR后进行测序及同源性分析。结果成功构建重组IFNβ刺激HepG2细胞差异表达基因的cDNA消减文库。文库扩增后，得到58个阳性克隆，进行菌落PCR分析，均得到200～1000bp插入片段。随机挑选其中35个插入片段测序，并通过生物信息学分析，结果共获得12种编码基因。结论应用SSH技术成功构建了IFNβ刺激HepG2细胞差异表达基因的cDNA消减文库，为进一步了解IFNβ在肝细胞内的免疫调节机制提供了依据。     

人类睾丸生精细胞凋亡相关基因TSARG3的克隆

目的：克隆人类睾丸生精细胞凋亡相关基因TSARG3。方法：从已获得的小鼠稳睾和正常睾丸对照中表达量有明显差异的表达序列标签片段（BE644537）入手，构建人同源表达序列标签重叠群，应用基因特异性引物和载体特异性引物，在睾丸cDNA文库的DN或进行巢式PCR扩增、测序，对测序结果进行生物信息学分析。结果：从睾丸cDNA文库中分离出人类睾丸凋亡相关基因的5’末端而获得全长cDNA,命名为TSARG3，GenBank登录号为AF419291（保密期为1年），同时应用相同方法克隆了该基因在小鼠中的同源基因，GenBank登录号为AF419292。结论：获得人类睾丸生精细胞凋亡相关基因TSARG3，该基因可能与人类睾丸生精细胞凋亡有关。     

快速检测四环素类抗生素的重组Lac Z基因酵母细胞的建立

以载体双表达的方式构建监测环境中四环素类抗生素污染的重组酵母细胞。在表达载体中,用3-磷酸甘油醛脱氢酶(GPD)启动子驱动四环素阻遏蛋白基因(TR)的表达,并使其与V5抗原表位编码基因相融合。在报告载体中,用四环素效应元件(TRE)调控的Lac Z作为报告基因。将两者转化于酵母细胞中,构建成四环素类抗生素调控的重组Lac Z基因酵母细胞。用不同浓度的四环素类抗生素和非四环素类抗生素对重组酵母细胞分别进行敏感度和特异度研究。结果表明四环素类抗生素与该重组酵母细胞报告基因表达水平有明显的剂量效应关系,说明对检测四环素类抗生素有较好的敏感性,而非四环素类药物与重组酵母细胞报告基因表达水平之间无明显的剂量效应关系,说明其检测具有良好的特异性。该重组基因酵母细胞可用于对环境四环素类抗生素污染程度的监测。     

自分泌运动因子受体基因及其干扰RNA表达载体的构建

目的 构建AMFR基因的表达载体和AMFRsiRNA表达载体，观察AMFRsiRNA对AMFR基因表达的影响。方法 采用PCR技术，扩增AMFR基因，并将其插入到带FLAG标签的真核表达载体上；利用RNAi技术，设计并合成了两条针对AMFR基因的siRNA，将其克隆到pSliencer 2.1-U6 neo表达载体上。将构建的AMFR基因表达载体和AMFRsiRNA表达载体共转染人胚肾细胞293T，通过Western blot实验了解AMFRsiRNA对AMFR基因表达的影响。结果 通过双酶切和测序鉴定表明，构建的AMFR基因表达载体和AMFRsiRNA表达载体序列正确；通过Western blot实验证明，构建的AMFRsiRNA能有效抑制AMFR基因的表达。结论 成功构建了AMFR基因表达载体和AMFRsiRNA表达载体，且表达的AMFRsiRNA能有效地抑制AMFR基因的表达。     

利用抑制消减杂交技术(SSH)研究与Ty21a免疫相关的基因

目的:利用抑制消减杂交技术（SSH）筛选与Ty21a免疫相关的新基因。方法:以Ty21a免疫小鼠的肠细胞为tester,以正常小鼠的肠细胞为diver,提取mRNA,反转录构建cDNA文库,利用SSH技术筛选Ty21a免疫相关的新基因。结果:通过SSH技术和cDNA微矩阵技术,发现了7条与GenBank中已公布的表达序列标签（expressed　sequence tag,EST）片段没有明显同源性,可能为Ty21a免疫相关的新基因。其中3条正在用RACE-PCR法进行获得全长cDNA的工作。结论:SSH技术是一种高效的差异基因筛选方法,基因表达谱芯片技术是高通量进行基因表达模式研究的方法,Ty21a可诱导多种免疫相关新基因的表达。     

Profile of Candida albicans-secreted aspartic proteinase elicited during vaginal infection

Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.     

Reaction of Candida albicans cells of different morphology index with monoclonal antibodies specific for the hyphal form.

Two monoclonal antibodies (MAbs), 3D9 with reported specificity for Candida albicans hyphae, and 3B7 with reported specificity for morphological forms of C. albicans found in vivo, were tested by indirect immunofluorescence with C. albicans cells that were grown in 12 different environments (four different culture media incubated at various temperatures) and whose cellular morphology was estimated in terms of morphology index (Mi). Both MAbs reacted strongly with cells with Mi greater than 3.0, i.e., with pseudohyphal and hyphal forms, but in Eagle's medium at 26 degrees C and in a modified Sabouraud's broth medium at 30 degrees C, some reactivity was also found with cells of lower Mi (i.e., yeast forms). Therefore, it was concluded that the hyphal phenotype and the epitopes reactive with the MAbs were co-expressed but that the epitopes could also be expressed independently of the hyphal phenotype. The results confirm the propensity of C. albicans for variation of its surface antigenic composition.     

Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants

The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.     

Gene expression in HL60 granulocytoids and human polymorphonuclear leukocytes exposed to Candida albicans

Candida albicans is an opportunistic human pathogen causing both superficial and disseminated diseases. It is a dimorphic fungus, switching between yeast and hyphal forms, depending on cues from its microenvironment. Hyphae play an important role in the pathogenesis of candidiasis. The host's response to Candida infection is multifaceted and includes the participation of granulocytes as key effector cells. The aim of this investigation was to study host gene expression during granulocyte-Candida interaction. Effector cells were generated by the granulocytic differentiation of HL60 cells. The resulting cell population was shown to be morphologically and functionally equivalent to granulocytes and is therefore referred to as HL60 granulocytoids for the purposes of this study. Gene expression profiles were determined 1 h after hosts were infected with C. albicans. Three Candida-granulocytoid ratios were chosen to reflect different degrees of HL60 granulocytoid inhibition of C. albicans. The data demonstrate that at the high pathogen-host ratio, C. albicans modulated the HL60 granulocytoid's response by downregulating the expression of known antimicrobial genes. In addition, looking at the expression of a large number of genes, not all of which have necessarily been implicated in candidastatic or candidacidal mechanisms, it has been possible to describe the physiological response of the HL60 granulocytoid to an infectious challenge with C. albicans. Finally, some of the observed changes in HL60 granulocytoid gene expression were investigated in freshly isolated human polymorphonuclear leukocytes infected with C. albicans. Similar changes were seen in these primary human cells, lending support to the validity of this model.     

Heterogeneous surface distribution of the fibrinogen-binding protein on Candida albicans.

As detected by indirect immunofluorescence and confocal microscopy, fibrinogen binding was heterogeneously distributed on the surface of Candida albicans. A low level of binding was generally observed homogeneously distributed on some yeast and most hyphal extensions of germ tubes. However, on most hyphal extensions, there were randomly distributed areas of increased expression, as revealed by patches of greater fluorescence intensity.