新疆克拉玛依地区皮肤利什曼病病原体SSU rRNA基因及k …

应用ＰＣＲ－ＳＳＣＰ银染技术，对皮肤利什曼病（ＣＬ）病人（Ｐ１７）的病原体ＳＳＵ ｒＲＮＡ基因及ｋＤＮＡ进行分析。将新疆克拉玛依地区皮肤利什曼病患者病变组织及有关利什曼原虫种株，Ｌ．ｉｎｆａｎｔｕｍ，Ｌ．ｔｒｏｐｉｃａ，Ｌ．ｄｏｎｏｖａｎｉ Ｘｉｎｊｉａｎｇ“７７１”株，分别抽提各标本的基因组ＤＮＡ和ｋＤＮＡ后，再用相应的引物，Ｒ２２２和Ｒ３３３ＰＣＲ扩增ＳＳＵ ｒＲＮＡ基因（３９７ｂｐ片段）和１     

巴西利什曼原虫激活蛋白激酶C受体的基因克隆化与序列分析

体外培养巴西利什曼原虫 (Lb ,Leishmaniabraziliensis)株无鞭毛体 ,常规方法提取制备基因组DNA。以公开发表的婴儿利什曼原虫 (Li,Leishmaniainfantum)的激活蛋白激酶C受体RACK (receptorforactivatedproteinCkinase)基因核苷酸序列为参照 ,设计并合成利什曼原虫RACK基因序列特异性的引物。以巴西利什曼原虫的基因组DNA为模板 ,利用多聚酶链反应 (PCR)技术 ,扩增获得巴西利什曼原虫RACK的全长编码基因。基因序列测定结果表明 ,巴西利什曼原虫RACK基因序列长度为 10 2 5bp ,开放读码框架由 939bp组成 ,编码产物为 312个氨基酸残基。获得的巴西利什曼原虫的RACK基因与报导的婴儿利什曼原虫的RACK基因编码产物的序列同源性达 99% (310 312 )。本研究克隆了巴西利什曼原虫的RACK基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行巴西利什曼原虫的基因疫苗研究奠定了基础。     

中国登革2型病毒分离株乳鼠致病性差异与基因变化关系的研究

本文报道１９８７年从广西病人血清中分离的登革２型（Ｄ２）病毒Ｄ２－４３株和１９８５年从海南病人血清中分离的Ｄ２－０４株乳鼠致病性的差异与基因变化的关系。结果表明Ｄ２－４３株对乳鼠致病，Ｄ２－０４株不致病。Ｄ２－４３株和Ｄ２－０４株Ｃ到ＮＳ１基因的读码框架基本相同，均由３３８１核苷酸组成，编码氨基酸总数１１２７。包含三个结构蛋白Ｃ．ＰｒＭ（Ｍ）、Ｅ和一个非结构蛋白ＮＳ１。该两株病毒核苷酸序列同源性为９３．８％，氨基酸序列的类似性为９１．３％。Ｃ和Ｅ基因同源性为９５．０％～９５．８％，ＮＳ１基因为９２．２％，Ｍ基因为８６．７％，两株之间核苷酸序列的主要差异是在Ｍ基因。两株病毒的Ｃ－ＮＳ１基因与国际参考毒株比较，４３株与ＪＡＭ株类侧性最高，其次是ＮＧＣ株，与Ｓ１株类似性最小。而０４株只有Ｃ、Ｅ和ＮＳ１与ＪＡＭ株最高，ＰｒＭ（Ｍ）都与ＮＧＣ株最高。４３株与０４株的Ｍ基因相比较，０４株的ＰｒＭ（Ｍ）基因的同源性低于４３株，说明两株与国际参考毒株比较，主要差异也在Ｍ基因，乳鼠致病性差异可能与Ｍ基因变化有关。     

登革4型病毒包膜蛋白基因的克隆和部分核苷酸序列测定

利用反转录多聚酶链反应（ＲＴ－ＰＣＲ）技术分离并克隆了登革４型０１株病毒的包膜蛋白（Ｅ）基因，并测定了部分核苷酸序列，发现该株与国外发表的一株相应区域核苷酸序列的同源性为９３．４％，由此推测出氨基酸序列的同源性为９２．１％。     

中国广西狂犬病毒野毒株（CGX89－1株）糖蛋白基因核酸序列测定和分析

本文报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％。核苷酸序列和巴斯德株（ＰＶ株），国际标准攻击毒株（ＣＶＳ株），中国狂犬病毒疫苗株（３ａＧ株）相比，同源性分别为８４．１％，８３．１％和８４．５％。其推导的氨基酸序列和ＰＶ株、ＣＶＳ株和３ａＧ株相比其同源性分别为９２．４％，８９．７％和８９．５％。ＣＧＸ８９－１株也具有３个Ｎ－糖基化位点，分别位于第３７位、１５７位和３１９位的氨基酸残基上。糖蛋白的膜外区部分重要抗原位点和ＰＶ株、ＣＶＳ株及３ａＧ株具有较高的一致性。     

5株呼吸道合胞病毒地方株F蛋白基因序列分析

目的呼吸道合胞病毒（ＲＳＶ）Ｆ蛋白是ＲＳＶ感染免疫中最重要的病毒蛋白，为了解我国ＲＳＶ地方株Ｆ蛋白的基因状况和变异特征，随机选取北京、广州、长春和河北四个地区具有不同流行特征的ＲＳＶ地方株（Ａ亚型）５株，进行ＲＳＶＦ蛋白全基因的核苷酸序列分析。方法以提取的病毒ｍＲＮＡ为模板进行ＲＴ－ＰＣＲ扩增、目的基因的克隆及序列测定，对地方株及原型株的序列进行比较分析。结果地方株Ｆ蛋白基因与原型株Ａ２株有很高的同源性，核苷酸全序列的同源性为９５．１％～９６．１％，氨基酸同源性为９６．７％～９７．４％。核苷酸有义突变率为２２．６％～２５．９％。３非编码区的核苷酸序列比蛋白编码区变异显著。河北地方株（Ｅ７３株）在３非编码区有６个核苷酸的插入。Ｆ２亚单位的氨基酸变异高于Ｆ１亚单位。在北京地方株（ＺＨＳ１３株）Ｆ１亚单位内，由具有中和能力单克隆抗体所识别的抗原表位区中存在一个氨基酸的变异。结论我国ＲＳＶ地方株与原型株之间的Ｆ蛋白基因尽管存在一定的变异，但仍有很高的同源性。地方株间Ｆ蛋白的核苷酸、氨基酸变异的位置及形式很相似，提示我国ＲＳＶ的不同流行特征可能并非由于Ｆ蛋白的基因变异所致。     

我国三株登革Ⅱ型病毒分离株包膜蛋白基因变化的…

本文报告了我国登革Ⅱ型病毒３个分离 株（０４、０５和４３株）包膜蛋白基因的核苷酸序列及推断的氨基酸序列。并通过美国ＭＡＣＡＷ微机程序对３株之间的核甘酸与氨基酸序列进行了比较分析，并分别与其它Ⅱ型毒株进行比较，发现０４、０５和４３株核苷酸序列同源性为９５．７６％～９５．９６％，它们之间无明显差别。氨基酸类似性为９２．１２％～９４．３０％，氨基酸类似性略低于核苷酸序列同源性。通过变化的核苷酸在三联密码     

不同基因型HCV膜区（E2）基因克隆及亲、疏水性分析

目的 研究丙型肝炎病毒（HCV）不同基因型膜区序列变异及其变异规律。方法 克隆不同基因型HCV的膜区基因,序列测定,核苷酸和氨基酸序列比较和分析。结果 不同基因型膜区基因相似性不同,从同一血清样本获得的不同克隆间,其同源性核苷酸大于99．2％,氨基酸大于99．9％。相同亚型核苷酸和氨基酸同源性大于80％。同型基因核苷酸和氨基酸同源性分别为（63．9－75．0）％和（64．9－72．8）％。异型基因同源性核苷酸为（59．4－67．0）％,氨基酸为（56．5－66．3）％。氨基酸序列变化在某些部位有一定保守性。不同基因HCV高变区（HVR）的亲水性和疏水性变化小于其下游3′端区域。结论 HCV基因变化可能有一定规律性。     

汉坦病毒A9株L片段部分核苷酸序列分析

目的 测定我国分离的汉坦病毒Ａ９株Ｌ片段的部分核苷酸序列。方法 用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术扩增我国分离的汉坦病毒Ａ９株部分Ｌ片段ｃＤＮＡ,测定ＰＣＲ产物的核苷酸序列。结果 ＰＣＲ扩增产物为Ｌ片段４００６ｎｔ－４４５４１ｎｔ（根据７６－１１８株序列）,和已发表的汉坦病毒Ｌ片段序列比较,Ａ９株和汉滩病毒７６－１１８株同源性最高,为８５．２％,和其他不同型别汉坦病毒代表株ＨＲ８０－３９、     

我国急性散发性戊型肝炎病毒部分核苷酸序列分析

用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）自深圳、长春、杭州等地４１份急性散发性戊型肝炎病人血清中获得２８株ＨＥＶｃＤＮＡ，对其中３株ＨＥＶｃＤＮＡ的ＯＲＦ２基因片段，用荧光法直接测定其核苷酸序列，并与戊型肝炎病毒墨西哥株（Ｍ）、缅甸株（Ｂ）和新疆流行株（ＣＨ１·１）进行了比较，结果该３株散发性ＨＥＶ与Ｍ株的核苷酸序列同源性分别为８０．２％、７９．９％和７９．４％；与Ｂ流行株的同源性为９５．５％、９３．９％和９５．１％；与散发株的同源性为９３．４％、９２．３％和９３．８％；与ＣＨ１·１的同源性为９７．０％、９６．５％和９５．９；表明该３株散发性ＨＥＶ与ＨＥＶ（Ｂ）和ＣＨ１·１的核酸序列同源性较高，可能属同一亚型。     

Rapid identification of causative species in patients with Old World leishmaniasis.

Conventional methods for the identification of species of Leishmania parasite causing infections have limitations. By using a DNA-based alternative, the present study tries to develop a new tool for this purpose. Thirty-three patients living in Marseilles (in the south of France) were suffering from visceral or cutaneous leishmaniasis. DNA of the parasite in clinical samples (bone marrow, peripheral blood, or skin) from these patients were amplified by PCR and were directly sequenced. The sequences observed were compared to these of 30 strains of the genus causing Old World leishmaniasis collected in Europe, Africa, or Asia. In the analysis of the sequences of the strains, two different sequence patterns for Leishmania infantum, one sequence for Leishmania donovani, one sequence for Leishmania major, two sequences for Leishmania tropica, and one sequence for Leishmania aethiopica were obtained. Four sequences were observed among the strains from the patients: one was similar to the sequence for the L. major strains, two were identical to the sequences for the L. infantum strains, and the last sequence was not observed within the strains but had a high degree of homology with the sequences of the L. infantum and L. donovani strains. The L. infantum strains from all immunocompetent patients had the same sequence. The L. infantum strains from immunodeficient patients suffering from visceral leishmaniasis had three different sequences. This fact might signify that some variants of L. infantum acquire pathogenicity exclusively in immunocompromised patients. To dispense with the sequencing step, a restriction assay with HaeIII was used. Some restriction patterns might support genetic exchanges in members of the genus Leishmania.     

用RAPD技术对利什曼原虫kDNA、nDNA的分析

目的 :用RAPD技术分析利什曼原虫的kDNA及nDNA。方法 :用 7种随机引物扩增L .infantum和L .donovaniGS2的kDNA和nDNA ,分析扩增结果并计算两虫种间kDNA和nDNA共享度F值。结果 :7种随机引物对两虫种的kDNA、nDNA均扩增出各自特有带型。L .infantum和L .donovaniGS2两虫种间kD NA、nDNA扩增片段的F值分别为 0 2 2 7和 0 2。结论 :kDNA和nDNA均可作为利什曼原虫的RAPD扩增模板 ,这 7种引物均可用于对L .infantum和L .donovaniGS2进行RAPD分析。该两虫种kDNA、nDNA的共享度较低 ,说明二者遗传距离较远。在进行RAPD分析时 ,提取全DNA (包括kDNA和nDNA)进行扩增即可     

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.     

A <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene from the pathogenic piscine hemoflagellate, <Emphasis Type="Italic">Cryptobia salmositica</Emphasis>

We report on the identification of a Cryptobia genomic DNA gene, predict it to encode a S-adenosylmethionine synthetase signature 1 motif and propose to name it S-adenosylmethionine synthetase (MAT). The open reading frame of MAT is 1,046 bp with 341 deduced amino acids. The MAT gene was identified using universal genome walking and Southern blot analysis revealed it to be a multi-copy gene. The S-adenosylmethionine synthetase of Cryptobia salmositica amino acid sequence is similar to those of other pathogenic kinetoplastids (Leishmania donovani 71%, Leishmania major 70%, Leishmania infantum 71%, Trypanosoma brucei 72%, Trypanosoma cruzi 70% and T. cruzi strain CL Brener 70%). The C. salmositica MAT has a conserved hexapeptide GAGDQG, which is widely found in bacteria, parasitic protozoans and also in humans. These suggest that MAT may have highly conserved functions such as regulation of gene expression and biosynthesis of a multitude of essential metabolites.     

Identification of 'Old World' Leishmania by DNA recombinant probes

Leishmania are usually identified by iso-enzyme analysis. This method works well, but there is a need for an additional, more simple, method of identification. Here we present data that show that in a Southern blot analysis, recombinant DNA probes in combination with certain restriction enzymes can differentiate between taxa of Leishmania. Probes based on clones selected from a L. infantum cDNA library gave characteristic patterns on Southern blots for reference strains of the different types of Leishmania found in Europe, Africa and Asia. Within the different taxa little or no variation was observed. Although the L. infantum derived probes showed a somewhat stronger hybridization for strains of the L. donovani complex, the signal obtained with most probes was satisfactory for L. major, L. aethiopica and L. tropica. Within the L. donovani complex none of the selected probes differentiated between isolates belonging to L. infantum, L. chagasi or L. donovani. Probes containing kinetoplast DNA showed considerable variation in hybridization within a taxon.     

Genetic variability within the species Leishmania infantum by RAPD. A lack of correlation with zymodeme structure

The infraspecific variability of the species Leishmania infantum is studied by using genetic markers generated by random amplified polymorphic DNA (RAPD). We have applied this technique, using 18 primers of arbitrary sequence, to 33 strains of the parasite belonging to 18 zymodemes isolated in different clinical forms and hosts. Other strains belonging to the species L. donovani, L. major, L. tropica and L. mexicana were used as a reference. The RAPD technique produced very different genetic profiles between L. infantum and L. major, L. tropica and L. mexicana with all primers used, whereas 11 of the 18 primers distinguished L. infantum strains from the species L. donovani. All primers except 1 (TAF 300), generated polymorphism in the L. infantum strains. The dendrograms constructed with the isoenzyme data and with RAPD are congruent in relation to the separation of the different species but show little agreement within the L. infantum species, reflecting the genetic heterogeneity of the strains belonging to one zymodeme. A geographical structuralisation is observed with two diverging groups that evolve independently whereas there is no relation between the genotype of the parasite and the host or between the former and the clinical form of the disease.     

Molecular epidemiology and population genetics in Leishmania

Polymorphic DNA sequences have been amplified using different PCR-based techniques and used for species identification, strain discrimination and population genetic studies in Leishmania. A PCR fingerprinting method that uses single non-specific primers generates species-specific banding patterns with some intraspecies variation. This approach can be used to identify Leishmania species and also to discriminate strains of different Leishmania species. Cultivation of the parasites is, however, mandatory. PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) in the ribosomal operon differentiates all Leishmania species, except members of the L. donovani and L. brasiliensis complexes. ITS-single-strand conformation polymorphism or ITS sequencing can detect strain specific-variation (except in L. infantum); culturing is not required. Species of Leishmania exhibit different degrees of genetic variation (L. tropica > L. aethiopica > L. major > L. donovani). Population analysis using co-dominant DNA markers developed by sequence-confirmed amplified region analysis revealed a primarily clonal structure in a L. donovani population from Sudan and suggested that occasional recombination events may occur in this population.     

Multilocus microsatellite typing as a new tool for discrimination of Leishmania infantum MON-1 strains

The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.     

Chromosome size polymorphisms of Leishmania donovani

A minimum of 22 chromosomes were found in all Leishmania donovani stocks examined by orthogonal field alternation gel electrophoresis (OFAGE). Chromosome sizes ranged from approximately 270 to 4000 kb. Certain chromosomes were polymorphic in size between stocks and chromosomes present in some stocks had no apparent equivalent in others. Specific polymorphisms were useful in distinguishing the subspecies L. d. donovani, L. d. infantum and L. d. chagasi and African L. donovani stocks but there were karotypic differences within these taxa. Radiolabelled DNA derived from whole chromosomes was hybridised to OFAGE Southern blots. Chromosome 1 of L. d. donovani was homologous to two larger chromosomes in all stocks. Chromosome 2 of certain L. d. chagasi and L. d. infantum stocks was homologous to both chromosomes 2 and 3 of L. d. donovani: this suggested that translocation between chromosomes may have contributed to the size polymorphisms. The smallest chromosome seen (270 kb) was unique to the African stock HU3. It was not homologous to small chromosomes in L. d. donovani, L. d. infantum or L. d. chagasi. The small chromosome did hybridise to two small chromosomes in another African stock, Khartoum, and to a large chromosome present in all stocks. The beta-tubulin gene was mapped to chromosomes 21/22, 13 and 7 with strongest hybridisation to 21/22. alpha-Tubulin was mapped to chromosomes 9. The alpha- and beta-tubulin arrangement was highly conserved.