问号钩体粘附侵袭相关基因特征分析

使用NCBI ,Swissprot/TrEMBL ,ProDom ,Pfam ,Tmpred ,SignalP ,ClustW等网络资源和软件 ,根据问号钩体黄疸出血型赖株粘附侵袭相关基因诠释结果 ,对mce ,invA ,mviN和atsE四个粘附侵袭相关基因编码蛋白的结构域、跨膜区域和信号肽等进行了详细分析 ,并使用Bioedit,Mega2软件进行氨基酸多重序列比较并绘制系统发生树。结果显示 ,mce和mviN为穿膜蛋白 ,invA和atsE为菌体内蛋白质 ;许多对哺乳动物和对植物致病的微生物具有mce ,invA ,mviN和atsE四个粘附侵袭相关基因 ,其表达的蛋白质在感染宿主过程中起重要作用 ,钩体的粘附侵袭相关蛋白与它们在一级结构上有较高相似性。据生物信息学结果推测 ,问号钩体黄疸出血型赖株粘附侵袭相关基因和钩体致病性间有密切关系 ,其编码蛋白在致病过程中可能起重要作用     

问号钩端螺旋体中一个新的OmpA家族蛋白抗原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体(L.interrogans)黄疸出血群赖型赖株中一个新的外膜蛋白(Omp)A家族基因LA0301,研究LA0301编码蛋白的抗原性和在15个钩端螺旋体(简称钩体)血清群代表株中的保守性,探讨其在疫苗研究中的意义。方法生物信息学软件分析预测LA0301的特征。构建原核表达重组体pQE31-LA0301,经IPTG诱导后用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法(Western blot)鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹检测其免疫反应性和在不同血清型钩体中的保守性。酶联免疫吸附试验(ELISA)和Western印迹检测兔抗钩体全菌血清中的LA0301编码蛋白的抗体。结果生物信息学预测结果显示,LA0301具有OmpA家族的结构域。克隆表达了重组质粒pQE31-LA0301,重组蛋白能刺激BALB/c小鼠产生特异性抗体,效价为1:32000。在兔抗钩体全菌血清中检测到特异的LA0301蛋白抗体,并在15个血清群的代表株钩体中均可检测到LA0301蛋白。结论LA0301蛋白是问号钩体中一个新的OmpA家族蛋白,具有良好的抗原性和保守性,并且能在钩体感染的过程中刺激机体产生相应的抗体。为进一步研究钩体新型疫苗候选基因奠定了基础。     

波摩那型钩端螺旋体外膜蛋白LipL32基因片段的克隆表达

目的　构建L3 2_pGEX_5x_2重组质粒 ,诱导表达重组钩端螺旋体外膜脂蛋白LipL3 2。方法　PCR获取编码LipL3 2的基因片段 ,构建重组克隆载体和表达载体 ,转化受体菌 ,诱导表达重组LipL3 2蛋白。将重组LipL3 2蛋白和钩体抗血清进行Western_blot。结果　扩增出约 750bp的LipL3 2成熟蛋白基因 ,LipL3 2基因插入pGEX_5x_2表达载体 ,表达产物谷胱甘肽S_转移酶 (GST ,2 6× 10 3)与LipL3 2蛋白的融合蛋白的相对分子质量约为 53× 10 3 ,与预期大小一致。Western_blot显示重组LipL3 2蛋白能与钩体抗血清特异结合。结论　LipL3 2蛋白能在大肠埃希菌中表达 ,重组LipL3 2蛋白具有免疫反应性     

秀丽隐杆线虫经福尔马林处理后上调表达基因的分析

秀丽隐杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前已经被广泛应用到生物对各种驱虫剂抗性机制的研究中。福尔马林被普遍用于鱼类寄生虫病的防治中,但是由于长期的应用,许多寄生虫对它产生了一定的抗药性。研究以秀丽隐杆线虫为研究对象,分析了它在福尔马林刺激下基因表达的改变情况。结果显示,经福尔马林处理后差异表达的有676个克隆,通过斑点杂交技术进一步筛选,对其中差异显著的161个克隆进行了测序分析。测序结果经BLAST分析发现:(1)细胞凋亡相关基因的表达发生上调,这些基因编码包括线粒体呼吸链相关蛋白、含TPR序列的蛋白SGT-1、热休克蛋白、氧化应激相关蛋白、胞吞过程相关蛋白、DNA复制和修复相关蛋白以及其他一些重要的凋亡相关蛋白;(2)编码重要的转录调节因子和信号转导相关蛋白如转录激活因子FosB/c-Fos、G蛋白、细胞周期蛋白B、钙结合蛋白、核小体装配蛋白NAP-1等的基因的表达发生上调;(3)能量代谢和蛋白质、脂肪、氨基酸代谢途径中的有些基因的表达也发生上调;(4)除了以上已知功能的基因外,还有一些未命名蛋白质基因。这说明福尔马林对秀丽隐杆线虫的影响除了诱导细胞凋亡之外,还影响其他细胞代谢活动的改变。实验为进一步研究生物体对福尔马林的抗性机制奠定了一定的基础。     

稻瘟菌WD重复功能域中SSR的变异及其对蛋白结构的影响

含WD重复功能域的蛋白能够参与信号传导、转录调控、RNA剪切、细胞的凋亡等多种功能,在病原菌与寄主植物蛋白互作的过程中扮演着重要的角色。本研究分析了稻瘟病菌基因组中94个WD功能域基因编码区和调控区中SSR的组成、分布,并检测了7个蛋白编码区中SSR的变异及其对蛋白二级结构的影响。结果表明,WD功能域基因的编码区和调控区中都含有大量的SSR,但是SSR在这些基因的外显子区、内含子区、5’一UTR和3’一UTR区中SSR的组成和分布均不相同;编码区中三碱基和六碱基SSR分布较多,这些SSR基序大都表现为GC含量较高和其所编码的亲水性氨基酸出现的频率远远高于疏水性氨基酸的特点。且检测的7个WD功能域基因的编码区中的SSR位点均具有丰富的多态性,通过Antheprot（DPM）软件预测发现：SSR的变异对蛋白的二级结构有一定影响。这暗示着SSR的变异对致病相关基因的变异起着十分重要的作用。     

大豆再生相关基因GmBIN2的克隆及生物信息学分析

根据前期实验获得的大豆Gm BIN2基因登录号,从大豆中克隆Gm BIN2基因的全长CDS序列,得到大豆Gm BIN2基因。对大豆再生相关基因Gm BIN2的启动子序列、氨基酸序列、编码的蛋白质结构、亲疏水性以及同源进化树进行分析,结果表明,大豆再生相关基因Gm BIN2编码区c DNA长度为1 125 bp,编码374个氨基酸,Gm BIN2编码的蛋白为亲水性蛋白;分析其蛋白功能结构域发现,Gm BIN2蛋白具有丝氨酸/苏氨酸激酶催化域,为PKc-like超家族成员;构建系统进化树发现其与野生大豆亲缘较近。本研究的实验结果有利于更加深入的研究Gm BIN2基因在大豆再生过程中的关键作用,为提高大豆再生效率提供依据。     

钩端螺旋体聚Beta羟基丁酸(PHB)生物合成途径分析

【目的】致病型问号钩端螺旋体(问号钩体, Leptospira interrogans)和腐生型双曲钩体(L. biflexa)能够大量合成菌体内贮藏物, 这可能是钩体在营养贫瘠环境中长时间存活的主要原因之一。本研究对钩体聚Beta羟基丁酸(PHB)贮藏物进行定性定量测定, 通过基因组分析补充定义PHB合成主要功能基因, 并采用分子生物学方法初步证明PHB合成途径的完整性, 为进一步研究PHB合成与钩体抗逆能力的关系奠定基础。【方法】采用脂类特异性尼罗红染色法和浓硫酸氧化-紫外分光光度计测定法, 对问号钩体和双曲钩体的PHB贮藏物进行定性定量测定; 采用生物信息学方法(BLAST和InterProscan/InterPro2Go), 通过同源性分析和功能结构域搜索寻找钩体基因组中的PHB合成相关基因; 最后采用克隆测序和定量RT-PCR技术检测相关基因表达情况, 初步验证生物信息学预测结果。【结果】尼罗红染色和氧化后比色定量实验证明钩体合成细菌常见贮藏物PHB, 问号钩体合成量为菌体干重的42%?45%, 双曲钩体合成量为64%?68%。尽管已公布的多个钩体基因组中均没有定义完整的PHB合成途径, 但本研究通过综合生物信息学分析, 在问号钩体和双曲钩体中鉴定了PHB合成途径的主要功能基因(phbC)。克隆测序和定量RT-PCR证实钩体转录表达大部分PHB合成相关基因(phbA/B/C), 说明钩体内该生物途径基本完整, 且部分高水平表达基因可能是钩体主要的PHB合成相关基因。【结论】问号钩体和双曲钩体均可合成PHB贮藏物, 且具有基本完整的PHB合成生物途径。     

捕获早老性痴呆基因

捕获早老性痴呆基因最近,美国国家变态反应及感染性疾病研究所的科学家建立了一种基因筛选系统,发现了与细胞凋亡有关的两个基因,这两个基因片断是从小鼠基因库中筛选出来的。其中一个基因片断编码蛋白可与重要信号分子钙结合,另一个与人类的早老性痴呆相关基因类似。...     

钩端螺旋体LA_1100蛋白膜定位研究

目的 研究LA_1100蛋白在钩端螺旋体中的膜定位并分析其在中国流行血清群中的保守性.方法 利用生物信息学软件对LA_1100的二级及三级结构进行分析,以Triton X-114抽提分离钩体细胞的各个组分,Westernblot及FACS方法验证LA_1100在钩端螺旋体中的膜定位.Western Blot和PCR在蛋白水平及核酸水平检测了其在13个中国流行血清群代表株中的保守性.结果 LA_1100的二级结构显示具有α-螺旋及β-折叠结构,进一步分析表明,此蛋白具有跨膜区.LA_1100单体结构具有典型的TolC结构域,三级结构模拟显示三聚体可形成孔状结构.膜定位显示,该蛋白定位于外膜.该基因的保守性分析结果表明,在中国13个流行代表株中有12株均检出该基因或该蛋白的特征性条带.结论 LA_1100为具有TolC结构域可以形成孔状结构的钩端螺旋体外膜蛋白,在中国流行株钩端螺旋体中保守.由于TolC蛋白与病原菌分泌致病因子密切相关,推测此蛋白可能与钩体感染宿主时粘附及致病相关,进一步对此蛋白进行研究有利于揭示钩体的致病机制.     

p53基因mdm2基因在肿瘤细胞中的相互调控作用

ｍｄｍ２（ｍｕｒｉｎｅ－ｄｏｕｂｌｅ ｍｉｎｕｔｅ２）最初是在一种可致瘤的小鼠在纤维细胞中发现的。该基因编码一种锌指蛋白,可与Ｐ５３蛋白酸性活化区结合,抑制Ｐ５３介导的转录活性阻止Ｐ５３诱导凋亡的作用。Ｐ５３基因是一种重要的抑癌基因,该基因编码一种ＤＮＡ结合磷蛋白,在调节细胞增殖和促进细胞凋亡过程中起着重要的作用。Ｐ５３也可调节ｍｄｍ２基因的表达。ｐ５３蛋白水平的升高级相应剌激ＭＤＭ２蛋白水平的升     

赖型钩端螺旋体外膜蛋白基因结构比较性研究

用ＰＣＲ方法扩增不同毒力赖型钩体ＯｍｐＬ１基因片段,进行序列测定,用相关软件比较分析核苷酸序列、蛋白质二级结构以及限制性内切酶谱,不同毒力赖型钩体能扩增出９６０ｂｐ的片段,非致病Ｐａｔｏｃ株未能扩出相应片段,中国赖型参考株ＯｍｐＬ１序列（ＧｅｎｅＢａｎｋ Ｎｏ．ＡＦ２５０３１８）与流感伤寒型相应序列比较有９８个核苷酸差异,同源性为８９．８％,二级结构预测和氨基酸疏水图显示变异主要发生在跨膜蛋白的膜     

Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

Leptospirosis is a globally important zoonotic diseasecaused by the pathogenic species of the spirochete genus,Leptospira including L. interrogans, L. kirschneri, L.noguchii, L. borgpetersenii, L. santarosai, L. weilii andetc. [1]. Pathogenic leptospires …     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Identification Leptospira santarosai serovar shermani specific sequences by suppression subtractive hybridization

In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar shermani. Suppression subtractive hybridization was employed to isolate DNA fragments present in pathogenic L. santarosai serovar shermani but absent in non-pathogenic L. biflexa serovar patoc. Analysis of 23 subtracted DNA clones revealed 25 gene fragments by BLASTX program. Eight clones showed similarity to transposase genes and three clones displayed homology with either translation or metabolism related genes. Four clones were similar to outer membrane protein, penicillin-binding protein, CreD-like protein and the protein of two-component signal transduction system, respectively. One clone had TPR repeat domain and five clones had significant similarity with hypothetical proteins of unknown functions. The remaining four clones exhibited no homology with any known genes. These results indicate that subtractive hybridization can successfully identify genes that are absent from the non-pathogenic Leptospira and provide a starting point for clarifying the differential genes expression between pathogenic and non-pathogenic Leptospira species.     

Etiological structure of icterohemorrhagic leptospirosis in gray rats (Rattus norvegicus)

In 93 Leptospira strains isolated from Norwegian rats serovar determination was made. As a result, leptospires circulating among Norwegian rats were found to belong mainly to serovar copenhageni, group Icterohaemorrhagiae, while leptospires of serovar icterohaemorrhagiae, even if occurring, were found only in the animals inhabiting pigsties. Leptospirosis epizooty among rats, caused by L. icterohaemorrhagiae, took its course independently of leptospirosis epizooty among mice, caused by L. hebdomadis, and simultaneously with it.     

An application of monoclonal antibodies to identification of leptospires of serogroup icterohaemorrhagiae found in China

For the purpose of improving the procedures of identification of leptospires, a set of 5 monoclonal antibodies with different serological reactivity against serovars of Leptospira interrogans Icterohaemorrhagiae serogroup isolated in China was developed. One hundred and eight strains isolated from epidemic fields in 5 provinces in southern China were distinctly identified into 4 serovars of Icterohaemorrhagiae serogroup by the monoclonal antibody procedure, i.e., 98 isolates were identified as serovar lai, 7 as icterohaemorrhagiae, 2 as copenhageni, and 1 as H2. Factor antiserum procedure was used at the same time as control for typing these strains and an identical result was obtained.     

Experimental lethal infection of Leptospira interrogans in mice treated with cyclophosphamide

After preadministration of cyclophosphamide (300 mg/kg), BALB/c mice were lethally infected with Leptospira interrogans serovar lai and a virulent strain of Leptospira interrogans serovar copenhageni, and leptospiral cells were detected in both kidneys of infected mice by indirect immunofluorescent assay. Nonpathogenic leptospirae, Leptospira biflexa serovar patoc, Leptonema illini, and an avirulent strain of L. interrogans serovar copenhageni, were not parasitic to the mice treated with cyclophosphamide. The cyclophosphamide-treated mice were protected from the homologous leptospiral infection by passive immunization with anti-leptospiral monoclonal antibody or with rabbit antiserum and by active immunization with lyophilized organisms or with protective antigen. The results of active immunization in mice treated with cyclophosphamide agreed well with those in nontreated hamsters, which were sensitive to the organisms. Furthermore, these experiments were reproducible with any lot of cyclophosphamide used. These results indicated that cyclophosphamide-treated mice can be used in the experimental infection of Leptospira in place of hamsters or guinea pigs.     

Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.