新生隐球菌荚膜相关基因CAP10启动序列的活性研究

目的 筛选具启动活性的新生隐球菌荚膜相关基因CAP10编码区上游5’侧翼序列（启动子序列），为进一步研究其调控机制打下基础。方法 利用氯霉素乙酰基转移酶（CAT）的编码基因为报告基因，构建了含不同长度的新生隐球菌荚膜相关基因CAP10编码区上游5’侧翼序列和报告基因的重组体，转化酵母，ELISA方法检测各转化子的CAT表达活性，并通过比较找到CAP10启动序列。结果 发现含CAP10编码区上游-303 bp、-390bp、-500bp、-951bp的转化子具有明显的CAT表达活性，而含-202bp、-102bp的转化子无明显的CAT表达活性。结论 CAP10上游5’侧翼端-303bp-0bp是具完整启动活性的最小单位。CAP10上游5’侧翼端-202bp--303bp内含CAP10启动所必需的序列。     

新生隐球菌荚膜基因CAP60与荧光蛋白融合表达系统的构建

荚膜是新生隐球菌的主要毒性因子 ,目前Ｃｈａｎｇ等已克隆了 4种荚膜基因 :ＣＡＰ5 9、ＣＡＰ6 4、ＣＡＰ6 0及ＣＡＰ10。已知这 4种基因对新生隐球菌的荚膜形成都是必须的 ,缺失任何一个基因都会使新生隐球菌变成无荚膜表型。搞清荚膜形成的任何一个生理、生化机制都将在新生隐球菌的临床诊断和治疗上带来突破性的进展。我们建立荧光蛋白作为标记与荚膜基因ＣＡＰ6 0融合表达 ,为进一步的研究创造条件。根据已知的ＣＡＰ6 0基因序列设计ＰＣＲ引物 ,上游为 5′ ＡＴＴＧＣＴＡＧＣＴＴＣＧＡ ＣＡＧＡＣＡＴＴＧＡＧＣＣＣＴ 3′ ,下游引…     

新生隐球菌耐药基因的检测

近年来 ,新生隐球菌性脑膜炎 (下称隐脑 )发病率呈日益上升的趋势 ,由于耐药菌株的增多应探讨新生隐球菌耐药基因及产生的原因。我们应用聚合酶链反应 ,对住院 2 1例“隐脑”病人 ,从脑脊液中分离出新生隐球菌 ,检测耐药基因。临床标本均从福州市三级医院住院“隐脑”病人的脑脊液中分离 ,且病人均经过两性霉素B、大扶康等治疗 6 5～ 80d。根据唑类抗真菌药物的作用靶酶羊毛甾醇 14α 去甲基化酶基因 ,设计的 1对引物P4 5 0 1和P4 5 0 2 。引物序列为 :P4 5 0 15′ ATGACTGATCAAGAAATYGCTAA 3′ ,P4 5 0 2 5′ TAACCTGGAGAA…     

新生隐球菌格鲁比变种国内菌株的多位点微卫星灶分型

目的 应用多位点微卫星灶分型（MLMT）对国内6个城市分离出的新生隐球菌格鲁比变种（Cryptococcus neoformans var.grubii）进行基因分型，了解该变种的国内基因型分布特征。方法 提取已鉴定的43株新生隐球菌格鲁比变种DNA，用PCR 对3个微卫星位点（CNG1, CNG2, CNG3）的基因片段进行扩增后测序.再计算每一菌株位点基序重复数（CNG1对应TA重复,CNG2对应GA重复,CNG3对应CAT重复）；据基序重复数判定各菌株的基因型。结果 所有43株菌中，MLMT-17型占83.72%, 该型在临床和环境的菌株中分别占86.67%, 70%。MLMT-39,-40是新发现的基因型。结论 在我国，MLMT-17是最常见的新生隐球菌格鲁比变种基因型，普遍存在于临床和环境菌株中。表明国内临床新生隐球菌病的感染菌株主要源于本土环境菌株。     

新型隐球菌野生株和荚膜缺陷株抗吞噬细胞吞噬作用的比较

目的　研究荚膜对新型隐球菌在巨噬细胞内外活力的影响 ,探讨荚膜在抗吞噬中的作用。方法　通过新型隐球菌标准野生株B35 0 1和荚膜缺陷株Cap6 0与小鼠巨噬细胞系J774细胞共孵育后 ,检测其出芽率、凋亡率 ,通过电镜观察隐球菌在J774细胞内的超微结构。结果　J774细胞对B35 0 1菌株的吞噬指数在 (5 .6 7± 1.2 9) %～ (8.76± 3.0 9) % ,对Cap6 0株的吞噬指数为 (31.6 2±12 .70 ) %～ (4 1.86± 9.74 ) % ;B35 0 1菌在J774细胞内的出芽率较高 ,可达 (4 6 .85± 6 .6 3) % ,而Cap6 0的胞内出芽率较低 ,仅达 (10 .73± 0 .39) % ;B35 0 1从共孵育时间达 4h后 ,出芽率开始下降 ,而Cap6 0从共孵育时间达 2h后 ,出芽率开始下降。流式细胞仪检测示J774细胞内、外的B35 0 1和Cap6 0菌株的凋亡率分别是 6 .4 %、4 .7%和 15 .4 %、9.6 %。结论　新型隐球菌可在J774巨噬细胞内存活并繁殖 ,荚膜不仅有抗吞噬的作用 ,而且有抗杀菌的作用     

新生隐球菌墨汁染色检查实验的改进

目的为消除生物安全隐患,让学生能安全高效地完成新生隐球菌墨汁染色检查实验。方法分别用10％EP醛或56℃水浴5rain对腹腔液、脑脊液中的新生隐球菌进行灭活处理,使其不再具有活性和致病性,镜下观察其形态的改变。结果用10％甲醛处理过的新生隐球菌,其荚膜形态已不能观察,而经56℃水浴灭活的新生隐球菌,其荚膜形态没有发生变化。结论新生隐球菌腹腔液或脑脊液采用56℃水浴5min即可灭活而不影响其荚膜形态,为学生安全高效地完成新生隐球菌墨汁染色检查实验提供了条件。     

广州地区患儿流感嗜血杆菌分离株荚膜基因分型的研究

目的用PCR方法进行流感嗜血杆菌荚膜编码基因分型,调查儿童急性呼吸道感染患儿流感嗜血杆菌的感染情况。方法以流感嗜血杆菌荚膜编码基因（bexA）和荚膜分型编码基因（Hi-a、Hi-b）作为靶基因设计引物,用PCR方法扩增标准菌株为ATCC9006、ATCC49247、EQA0609的3个编码基因并测序,在此基础上对临床分离的43株流感嗜血杆菌进行荚膜基因分型研究。结果PCR结果显示标准菌株ATCC49247未扩增出bexA编码基因,ATCC9006扩增出bexA和Hi-a编码基因,EQA0609扩增出为bexA和Hi-b编码基因,并且PCR产物序列与GenBank公布的序列一致性高。临床分离菌株Hi未扩增出bexA、Hi-a及Hi-b编码基因。结论本实验研究表明广州地区儿童急性呼吸道感染患儿所分离的流感嗜血杆菌主要是不可分型的无荚膜菌株。     

国内110株新生隐球菌临床株变种、基因型和交配型分析

目的 对国内部分地区的新生隐球菌(Cryptococcus neoformans)临床株进行分子流行病学调查,分析其变种、基因型和交配型的构成和分布.方法 (1)PCR指纹分型法:以野生型噬菌体M13中针对小卫星DNA的核心序列为单引物对模板进行PCR扩增,将所有受试菌株鉴定到8种主要基因型水平.(2)利用变种和交配型特异性引物扩增分型法,区分格鲁比变种(C.neoformans var.grubii)、新生变种(C.neoformans var.neoformans)和格特变种(C.neoformans var.gattii),同时鉴定α和a交配型.结果 110株临床株中,98株(89.1%)为格鲁比变种,均为VNI基因型和α交配型;9株(8.2%)格特变种,包括VGⅠ基因型、α交配型8株(7.3%)和VGⅡ基因型、α交配型1株(0.9%);2株(1.8%)为AD杂合体,VNⅢ基因型,-/α和α/-交配型各1株;1株(0.9%)为新生变种,VNⅣ基因型和a交配型.结论 我国新生隐球菌临床株包含3个变种和AD杂合体.与国外情况比较,相似的是国内临床株中绝大部分为α交配型菌株,且格鲁比变种中的VNⅠ基因型占了其中的大部分;但未发现VNⅡ、VGⅢ和VGⅣ基因型菌株.     

新生隐球菌格鲁比变种临床株与环境株微卫星基因型的小鼠毒力实验研究

目的 了解巴西新生隐球菌格鲁比变种(C. neoformans var.grubü)多位点微卫星定型(multilocus microsatllite typing,MLMT)在临床和环境中的分布特点,探讨不同微卫星型的新生隐球菌格鲁比变种临床株和环境株的毒力差异.方法 根据多位点微卫星分型技术对分离自巴西的40株(17株临床株和23株环境株)新生隐球菌格鲁比变种进行分型,筛选出临床株及环境株中分布占优势的菌株,应用小鼠毒力试验分别对其进行毒力检测,通过发病情况及病理切片比较毒力差异.结果 40株格鲁比变种共鉴定出11种微卫星型,临床株中的优势菌株为MLMT-13型,共9株,占临床株的52.9%(9/17),环境株中的优势菌株为MLMT-36型,共10株,占环境株的43.5%(10/23);小鼠毒力试验结果显示MLMT-13型感染小鼠后发病率为100%,而MLMT-36型发病率为7.5%,且病理结果也存在明显差异.结论 格鲁比变种在从环境迁徙到宿主的过程中,毒力随环境的变化发生了改变,该变化也可在其微卫星重复序列上体现出来.该实验证明新生隐球菌格鲁比变种的分布及来源与微卫星型之间存在相关性,临床株MLMT-13型和环境株MLMT-36型之间存在着显著毒力差异.

Abstract：

Objective To investigate the genetic relation between Cryptococcus neoformans var.the clinical strains in MLMT - 13 genotype and the environmental strains in MLMT - 36 genotype. Methods Multilocus microsatellite typing (MLMT) method was applied for the genotype analysis in our study.Through this method, we recognized two genotypes that distinguish a majority of clinical and environmental strains. In order to compare virulence between the two types, we chose to infect BALB/c mice (6 weeks,female) with 9 MLMT-13 strains and 10 MLMT-36 strains intravenously. Results Forty( 17 clinical and 23 environmental isolates) were analyzed. Of 17 clinical strains, 9 belonged to a major type of MLMT-13 (52.9%). They were mainly isolated from clinical specimens. About 43.5% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. The mortality rate and pathological changes of the above mice were observed during two months after injection. The results showed that the mortality rate of mice infected with MLMT-13 strains was 100%, while the mortality rate with MLMT-36 strains was 7. 5%. The pathological sections showed that lesions of MLMT-13 infected mice appeared in the brain, lungs, liver and kidneys, while the lesions of MLMT-36 infected mice only appeared in the brain. Most brains of MLMT-13 infected mice were distorted,and both the number and size of lesions in such brains were much larger than those of MLMT-36 infected mice. Conclusion Our study illustrated the virulent difference between MLMT-13 and MLMT-36, which are isolated from patients and environment respectively. The results inferred that some genetic changes, such ss microsatellite repeats, might occur between environmental and clinical isolates through their environmental adaptation progress.     

应用完整外显子2/3序列解决HLA-DRB1座位基因分型的歧义结果

背景：人类白细胞抗原基因测序分型中,当等位基因的差异碱基位于测序范围之外或不同等位基因对的杂合序列相同时,无法得到清晰的结果。 目的：通过完整外显子2/3序列的测定,解决常规HLA-DRB1基因分型中的高比例歧义结果。 方法：初次分型采用常规的测序方法检测320份样本的HLA-DRB1外显子2第一高变区以外的序列,测序反应设置codon86。后期采用一次性扩增外显子2/3,测序反应针对外显子2设置组特异性引物：DRB1*04/07/09为一组,其它基因家族为一组,设置conden86,对初次分型后为歧义结果的样本重新分型。 结果与结论：初次分型有180份样本为歧义结果,占总样本数的56.25 %。其中A类为差异碱基位于测序范围之外,共114例;B类为等位基因对的杂合序列相同,共17例;C类为两种情况同时存在,有49例。3种类别的歧义等位基因数分别为119个、34个、98个,占等位基因总数的33.06%、9.44 %、27.22%。完整外显子2/3序列的测定使歧义结果比例从56.25%下降到14.37%,其中A类103例、B类8例、C类23例样本的等位基因得到确认。此次研究中发现了一个新等位基因,与跟它最相近的等位基因DRB1*110101相比,其外显子3的第381位碱基G>T,导致第98位氨基酸AAG(赖氨酸 Lys)>AAT(天冬酰氨 Asn)。序列已提交Genbank,编号HM807583,2010-08被世界卫生组织HLA因子命名委员会命名为HLA-DRB1*1197(编号HWS10010999)。提示,完整外显子2/3序列的测定能大幅降低歧义分型结果的比例。     

Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages.

Our previous studies have shown that unstimulated alveolar macrophages (AM) play a predominant role as antigen-presenting cells in Cryptococcus neoformans infections, while the function as effector cells seems to be of minor relevance. The present study focuses on the role of encapsulation of C. neoformans on fungicidal activity and the antigen presentation process of AM. Fungicidal activity in unstimulated AM occurs to a higher degree when the acapsular strain is employed, but this is impaired compared with other natural effectors, such as peripheral blood monocytes (PBM) and polymorphonuclear (PMN) cells. Cryptococcus-laden AM also induce a higher proliferative response in autologous CD4+ lymphocytes when the acapsular strain is used compared with encapsulated yeast. The enhanced blastogenic response is, in part, ascribed to an augmented IL-2 production by T cells. In addition, higher levels of interferon-gamma (IFN-gamma), but not IL-4, are produced by the responding T cells, when the acapsular strain is used compared with the encapsulated yeast. Moreover, IFN-gamma is able to induce fungicidal activity in AM against the encapsulated yeast and augments killing activity of the acapsular strain. This phenomenon is not mediated by nitric oxide production, but is correlated with an enhancement of fungicidal activity of cytoplasmic cationic proteases. We speculate that encapsulation of C. neoformans could down-regulate the development of the immune response mediated by Cryptococcus-laden AM at lung level.     

Variability of karyotypes and RAPD types in genetically related strains of Cryptococcus neoformans

Variation in karyotypes and RAPD patterns of genetically related strains of Cryptococcus neoformans were analyzed. Capsular and filamentous mutants usually differ in their karyotypes from wild-types, but the RAPD patterns were found to be similar. Karyotype differences were observed in most heterothallic matings, but RAPD patterns remained identical. After self-sporulation of a diploid strain, minor chromosomal length polymorphism and minor changes in the RAPD types occurred. Three mechanisms, either alone or in combination, may in varying degrees contribute to the karyotype variation of C. neoformans: (1) mitotically induced changes; (2) karyotype changes as a result of meiotic recombination, and (3) mutagen-induced changes. The present data do not support the meiotic maintenance hypothesis, which claims that the amount of CLP generated is inversely proportional to the frequency of meiosis. Received: 24 February / 26 June 1997     

CD59真核表达载体的构建及其对Jurkat细胞增殖的影响

目的构建pIRES-CD59融合基因真核表达载体,探讨重组CD59在Jurkat细胞增殖中的作用。方法 RT-PCR法选择性扩增编码CD59的基因片段,克隆入pIRES真核表达质粒。脂质体法将重组载体转染Jurkat细胞,通过G418筛选获得稳定表达CD59的细胞克隆,利用RT-PCR和Western blot检测细胞中CD59的表达,通过MTT法检测Jurkat细胞的增殖。结果经酶切和测序鉴定表明携带CD59基因的重组质粒构建成功,RT-PCR和Western blot结果显示转染Jurkat细胞的CD59基因高表达,MTT实验显示转染CD59重组质粒的Jurkat细胞增殖速度明显快于对照组(P<0.05)。结论 pIRES-CD59真核表达载体在转染细胞Jurkat中可高表达CD59分子,并可影响Jurkat细胞的增殖,为研究CD59的生物学作用奠定了基础。     

Deletion of the CAP10 gene of Cryptococcus neoformans results in a pleiotropic phenotype with changes in expression of virulence factors

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is an important virulence factor for this yeast-like fungus. Previously, we had shown that disruption of the CAP10 gene, encoding a putative xylosyltransferase, results in mutant cells that lack most of the capsular polysaccharides on the cell surface, but do not show a typical acapsular phenotype. In contrast to the acapsular cap59 mutant, cap10 did not induce maturation of dendritic cells when exposed to components of the immune system. In order to gain further insight into the causes of this phenotype displayed by the cap10 mutant, we performed a more in-depth phenotypic analysis of the cell wall and surface structures of this mutant compared to the wild type strain and acapsular mutant cap59. Moreover, we analyzed the cap10 mutant and the wild type strain for differential gene expression of, amongst others, enzymes that are involved in biogenesis of cell wall and capsule components. We conclude that a mutation in the CAP10 gene results in a pleiotropic phenotype with effects on different cellular processes affecting, amongst others, cell size, expression of virulence factors and size of extracellular vesicles.     

Influence of gender and age on course of infection and cytokine responses in mice with disseminated Cryptococcus neoformans infection

Objective   To study the influence of gender and age on the course of infection and the cytokine response in a murine model of disseminated cryptococcosis.
Methods   The course of the infection (survival and fungal load in blood and tissues) as well as pro-inflammatory and anti-inflammatory cytokine responses in plasma and organs were compared according to gender and age in outbred mice previously infected with Cryptococcus neoformans NIH52D.
Results   Although survival and fungal load were similar in male and female mice, the expression of all cytokines in plasma and of tumour necrosis factor- α and interferon- γ in spleen was significantly increased in female mice compared to male mice in two independent experiments. Young male mice had a significantly shortened survival, were significantly more infected and had predominant tumour necrosis factor- α and interferon- γ responses in comparison with older male mice.
Conclusion   Host factors should be taken into account when studying the immune response to experimental C. neoformans infection. Our data support epidemiological and clinical data showing differences in susceptibility to cryptococcosis according to gender and age.     

Eosinophils elicit proliferation of naive and fungal-specific cells in vivo so enhancing a T helper type 1 cytokine profile in favour of a protective immune response against Cryptococcus neoformans infection

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, because as in healthy humans, rats can effectively contain cryptococcal infection. Moreover, it has been shown that eosinophils are components of the immune response to C. neoformans infections. In a previous in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, thereby triggering their activation, as indicated by the up‐regulation of MHC and co‐stimulatory molecules and the increase in interleukin‐12, tumour necrosis factor‐α and interferon‐γ production. Furthermore, this work demonstrated that C. neoformans‐specific CD4⁺ and CD8⁺ T lymphocytes cultured with these activated C. neoformans‐pulsed eosinophils proliferated, and produced important amounts of T helper type 1 (Th1) cytokines in the absence of Th2 cytokine synthesis. In the present in vivo study, we have shown that C. neoformans‐pulsed eosinophils are also able to migrate into lymphoid organs to present C. neoformans antigens, thereby priming naive and re‐stimulating infected rats to induce T‐cell and B‐cell responses against infection with the fungus. Furthermore, the antigen‐specific immune response induced by C. neoformans‐pulsed eosinophils, which is characterized by the development of a Th1 microenvironment with increased levels of NO synthesis and C. neoformans‐specific immunoglobulin production, was demonstrated to be able to protect rats against subsequent infection with fungus. In summary, the present work demonstrates that eosinophils act as antigen‐presenting cells for the fungal antigen, hence initiating and modulating a C. neoformans‐specific immune response. Finally, we suggest that C. neoformans‐loaded eosinophils might participate in the protective immune response against these fungi.     

CD59-CD2对T细胞信号转导的作用

目的将构建的CD59-linker-CD2融合基因真核表达载体在Jurkat细胞中稳定表达,研究CD59与CD2分子在T细胞信号转导中的相互作用,探讨CD59向T细胞内传递信号的相关机制。方法酶切鉴定构建的pIRES-CD59-linker-CD2融合基因真核表达载体,脂质体法转染Jurkat细胞,G418筛选建立稳定转染细胞系,免疫酶法检测转染细胞表面CD59的表达,Western blot检测转染细胞中CD59蛋白的表达;用CD59单克隆抗体作用于各组Jurkat细胞,使用激光共聚焦显微镜检测细胞浆内的Ca2+,ELISA检测细胞上清中白介素2(IL-2)的变化,比较各组差异。结果经酶切鉴定证实携带CD59-linker-CD2融合基因的重组质粒扩增成功;免疫酶法和Western blot证实CD59在转染细胞中稳定表达,且转染组L-Jurkat细胞比正常组Ju-rkat细胞CD59表达量增加,二者比较其差别有统计学意义(P<0.05);与正常细胞相比,胞浆内Ca2+和IL-2在转染细胞中均高表达,两组细胞比较其差别有统计学意义(P<0.05)。结论 pIRES-CD59-linker-CD2融合基因真核表达载体可在Jurkat细胞中稳定表达,CD59mAb交联刺激后,CD59可通过CD2向细胞内传递信号,引起细胞内信号分子的一系列变化,为研究CD59与CD2在T细胞信号转导中的协同作用及进一步探讨CD59向T细胞内传递信号的机制奠定基础。     

人突变CD59基因在CHO细胞的表达、检测及生物活性研究

目的：分别构建两个高效表达人突变CD59的CHO细胞真核表达系统，探讨突变CD59基因表达蛋白糖基化前后抗补体活性的变化，为在基因水平阐明糖尿病血管增殖症的发病机理奠定基础。方法：将两种含不同突变基因的人CD59全长cDNA序列（HM7、HM8）的重组pALTER质粒运用阳离子脂质体导入法共转染CHO细胞，用含有400μg/ml新霉素类似物（G418）的F12培养基筛选出阳性表达克隆，应用荧光免疫组化（FIH）检测CD59在CHO细胞表面的表达。经染料释放试验检测突变CD59糖基化前后抗补体活性。结果：运用阳离子脂质体导入法将重组pALTER质粒与pcDNA3质粒共转染CHO，成功筛选出的阳性克隆经FIH证明突变CD59可在CHO细胞表达。染料释放法证实两种突变CD59均具有抗补体活性，且在高糖环境下易糖基化，糖基化后抗补体活性明显降低。结论：建立了两个高效表达突变CD59的真核表达系统，获得阳性克隆细胞株。初步研究证实突变CD59具有抗补体活性，糖基化后抗补体活性明显降低，为单克隆抗体的制备奠定了基础。     

CD59融合蛋白表达载体构建及其在大肠杆菌中的表达

探索ＧＰＩ－锚固蛋白的体外表达，获得具有生物学活性的可溶性游离ＣＤ５９分子。方法通过ＰＣＲ选择性扩增编码成熟ＣＤ５９氨基酸序列的基因片段，并将之克隆入ＰｉｎＰｏｉｎｔＸａ－３质粒中。ＩＰＴＧ诱导融合蛋白表达，产物经亲和素树脂亲和层析。在丛外微量反应性溶血抑制实验中检测重组ＣＤ５９纯化样品的生物学活性。