The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences

An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.     

3株禽滑液囊支原体分离株致病性的比较和评价

【背景】禽滑液囊支原体是感染鸡的一种重要病原体,给我国的家禽养殖业带来了严重的经济损失。【目的】评价从病鸡中分离到的3株禽滑液囊支原体的致病性,以期丰富不同区域来源的分离株对鸡致病性的认识。【方法】分别用分离株CHN-WF224-2016、CHN-BZJ2-2015、CHN-JNB19-2016感染商品肉鸡,并在攻毒后第10天和21天进行实验室解剖,通过气囊和足垫的临床病变、血清学检测结果及气管粘膜病理形态学改变来比较评价分离株的致病力。【结果】CHN-BZJ2-2015和CHN-WF224-2016分离株能引起明显足垫肿胀和关节滑膜炎,其中CHN-BZJ2-2015分离株致病力最强,感染21 d后的血清学检测和气管粘膜厚度与CHN-JNB19-2016分离株和MS-H疫苗株比较差异显著(P<0.05)。【结论】本研究表明我国目前流行的禽滑液囊支原体菌株其致病力存在明显差异,强调养殖场要积极控制和预防禽滑液囊支原体感染的重要性,同时为今后该疫苗的研发积累了实验数据。     

滑液支原体NADH氧化酶的酶学活性及亚细胞定位研究

【背景】许多研究表明,支原体的NADH氧化酶(NADH oxidase,NOX)不仅在胞浆中发挥生物酶学功能,也存在于细胞膜上发挥黏附宿主细胞功能。【目的】对滑液支原体(Mycoplasma synoviae,MS)的NOX进行酶学活性及亚细胞定位研究,分析其在MS致病过程中的潜在作用。【方法】对MS的NOX蛋白进行原核表达、纯化,然后对重组MSNOX (rMSNOX)的酶学活性及影响酶活的条件进行研究,测定其酶比活力、米氏常数及最大反应速率,接着用MS阳性血清及制备的rMSNOX兔多克隆抗体,分别与rMSNOX蛋白及MS全菌、膜蛋白和胞浆蛋白进行Western blotting反应,鉴定rMSNOX的免疫原性及其在MS中的分布情况。【结果】在大肠杆菌BL21(DE3)中成功表达rMSNOX蛋白,相对分子质量约为53 kD,并获得纯化的rMSNOX蛋白;酶活测定显示rMSNOX蛋白的酶比活力为14.17IU/mg,最适酶促温度为37℃,最适pH为7.5,双倒数法求得rMSNOX的最大反应速率V_max为21.8μmol/(L·min),米氏常数K_m     

滑液支原体醛缩酶的亚细胞定位及免疫原性

【背景】滑液支原体(Mycoplasma synoviae,MS)感染能够引起鸡和火鸡的气囊炎、关节渗出性的滑液囊膜及腱鞘滑膜炎等。有研究表明,许多支原体中与代谢相关的酶类不仅分布在细胞质,也分布于细胞膜表面,通过结合宿主细胞的胞外基质蛋白,协助病原菌黏附入侵宿主细胞。已有报道牛支原体(Mycoplasma bovis)和鸡毒支原体(Mycoplasma gallisepticum)的醛缩酶(Fructose-bisphosphate aldolase,FBA)分布在膜蛋白和胞浆蛋白中,而MS的FBA蛋白还未见相关研究。【目的】对MSFBA蛋白进行生物信息学分析、原核表达、免疫原性分析及亚细胞定位检测,为进一步探索MS代谢相关酶类的生物学功能奠定基础。【方法】通过分析软件PSORTb、SignalP 4.1 Server和TMHMM Server在线预测MSFBA的亚细胞定位、信号肽及跨膜区,并用BLASTn和MEGA 5.0进行同源比对及进化树分析;通过Overlap PCR点突变扩增MS的fba全基因序列,连接表达载体pET-28a(+)并进行原核表达及纯化,获得纯化后的重组MSFBA(rMSFBA)蛋白;用MS阳性血清进行Westernblot鉴定rMSFBA的免疫原性;用纯化的rMSFBA蛋白制备兔多克隆抗体,与MS全菌蛋白、膜蛋白及胞浆蛋白进行Western blot分析,同时对MS全菌进行悬浮免疫荧光分析,检测MSFBA的膜定位情况。【结果】生物信息分析预测MSFBA分布在细胞质中,无信号肽,无跨膜区,在MS种内相似性高达99%,与其他种属FBA相似性在61%-78%之间,进化树显示其与牛鼻支原体(Mycoplasma bovirhinis)、仓鼠支原体(Mycoplasm acricetuli)等的FBA蛋白进化关系较近,与精氨酸支原体(Mycoplasma arginini)、人型支原体(Mycoplasma hominis)等的FBA蛋白进化关系较远;表达rMSFBA蛋白并纯化,经测定其相对分子质量大小约为33kD;rMSFBA能与MS阳性鸡血清特异性结合,证实其具有较好的免疫原性;Western blot显示抗rMSFBA的兔血清能与MS全菌蛋白和胞浆蛋白反应,而与膜蛋白不反应,说明MSFBA蛋白分布于胞浆中;悬浮免疫荧光实验证实MS的细胞膜上未见FBA蛋白分布。【结论】首次报道了滑液支原体的FBA蛋白是一个高度保守的免疫原性蛋白,主要分布在细胞质中,该结果为进一步研究MSFBA蛋白的生物学功能提供了分子基础。     

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.     

Elongation factor (EF-Tu) gene probe detects polymorphism in Mycoplasma strains

Abstract Polymorphism in mycoplasma strains was observed by Southern blot hybridization of the digested mycoplasma DNAs with the elongation factor (EF-Tu) gene tuf of Escherichia coli . The hybridization patterns revealed genotypic heterogeneity among Mycoplasma gallisepticum strains and a remarkable degree of homogeneity among Mucoplasma pneumoniae strains isolated from pneumonia patients. The distinction among M. gallisepticum strain clusters achieved by the tuf gene probe corresponded exactly with that obtained with the rRNA gene probe pMC5. The tuf gene probe may thus be added as another effective tool in the taxonomy of Mollicutes and in epidemiological surveys of mycoplasma infections.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.     

山东地区16株H9N2亚型禽流感病毒HA基因的序列分析

为了解H9N2亚型禽流感病毒(AIV)山东分离株的遗传变异情况,采用RT-PCR技术对16株从山东不同地区分离的H9N2亚型禽流感病毒的HA基因进行扩增、克隆和测序,并对所获得的HA全序列进行同源性和遗传进化分析。结果显示,16个分离株的裂解位点均为RSSR↓GLF,符合低致病性禽流感病毒的分子特征;有7～9个潜在糖基化位点;受体结合位点除198位有变异,其他位点均较保守;234位氨基酸均为L,具有与哺乳动物唾液酸α,2-6受体结合的特征;16个分离株HA基因核苷酸及氨基酸序列同源性分别为96.3%～99.9%和97.1%～99.6%;16个分离株同属于欧亚分支中的A/Duck/Hong Kong/Y280/97亚群。     

Vsp antigens and vsp-related DNA sequences in field isolates of Mycoplasma bovis

The expression of the 1E5 epitope which is common to the three characterized variable lipoproteins VspA, VspB and VspC of Mycoplasma bovis type strain PG45 and the presence of vsp gene DNA sequences were assessed in field isolates randomly collected from cattle showing clinical manifestations due to M. bovis infection. Among 250 isolates tested, only four failed to react with mAb 1E5. Southern blot analysis of these four isolates and of 20 isolates expressing the 1E5 epitope were performed using synthetic oligonucleotide probes corresponding to a sequence located in the Vsp signal peptide coding region common to all known Vsp products or to selected regions of previously characterized vsp genes, vspA, vspE and vspF. The results demonstrate the presence of multiple vsp-related DNA sequences in all M. bovis field isolates tested and indicate that the vsp repertoire varies in size and composition among isolates.     

Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam

Moringa oleifera is an important multipurpose tree introduced to Africa from India at the turn of this century. Despite limited knowledge of the levels of genetic diversity and relatedness of introduced populations, their utilization as a source of seed for planting is widespread. In order to facilitate reasoned scientific decisions on its management and conservation and prepare for a selective breeding programme, genetic analysis of seven populations was performed using amplified fragment length polymorphism (AFLP) markers. The four pairs of AFLP primers ( Pst I/ Mse I) generated a total of 236 amplification products of which 157 (66.5%) were polymorphic between or within populations. Analysis of molecular variance ( AMOVA ) revealed significant differences between regions and populations, even though outcrossing perennial plants are expected to maintain most variation within populations. A phenetic tree illustrating relationships between populations suggested at least two sources of germplasm introductions to Kenya. The high levels of population differentiation detected suggest that provenance source is an important factor in the conservation and exploitation of M. oleifera genetic resources.     

Molecular genetic basis of tapetoretinal degeneration

The review considers tapetoretinal degeneration (TD), a severe incurable disease occurring at a frequency of 1 per 3500–5000 people. TD is most commonly caused by mutations of the genes for rhodopsin (RHO), peripherin (RDS), and retinol acetyltransferase (RPE65). Since pigmentary degeneration strongly correlates with mutations of these genes, it is possible to develop approaches to DNA diagnosis of hereditary retinal dystrophies, which are common in practical ophthalmology, and to exactly, rather than probabilistically, evaluate its risk. Molecular analysis of the TD-associated changes in the genes that ensure the proper function of photoreceptors and the retinal pigment epithelium will provide for a better understanding of the physiological and pathological processes occurring in the retina, as well as for the development of pathogenetic therapy in TD.     

猪肺炎支原体p97 C末端基因重组腺病毒的构建及其免疫效果

摘要：【目的】构建携猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）黏附因子p97 C末端基因的重组腺病毒并研究其诱导小鼠产生的免疫反应,为研制新型Mhp疫苗奠定基础。【方法】从Mhp基因组中扩增p97 C末端基因,并将其克隆到穿梭载体pShuttle-CMV中,该重组穿梭质粒经Pme I线性化后电转化到BJ5183-AD-1细胞中进行同源重组获得重组腺病毒DNA,纯化后的重组腺病毒质粒经Pac I酶切线性化后转染AD293细胞以获得重组腺病毒。对该重组腺病毒进行RT-PCR、间接     

Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.     

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

Abstract The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of M. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.     

Structural basis for restriction-site polymorphism at the albumin locus in inbred strains of rats

Two types of variant EcoRI restriction enzyme patterns of albumin-gene DNA fragments are found in different inbred strains of rats and reflect allelic polymorphism. The structural basis of the two allelic forms has been analyzed by mapping the EcoRI fragments using cloned albumin cDNA probes corresponding to the 5 or 3 end of the rat albumin mRNA and different genomic subclones. Additional restriction fragment length polymorphism has been detected using the restriction endonucleases HindIII and MspI. The results suggest that the two allelic variants differ from each other by multiple cleavage-site variations (base-pair substitutions) and by an insertion or deletion of DNA sequences. An extensive DNA sequence variation appears to exist between the two forms of the albumin gene; we have estimated that as much as 4% of the nucleotides in this region varied between the two alleles. All of this genetic variation is found in the intervening sequences of the gene and has no phenotypic manifestation.This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (Contract 7940 222) and the Association pour le développement de la Recherche sur le Cancer (Contract 6109). A. Gal was supported by a fellowship from the Institut National de la Santé et de la Recherche Médicale.On leave of absence from Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.     

Genomic variations of Mycoplasma capricolum subsp. capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size range of 40-500 bp. The similarity between individual AFLP profiles, calculated by Jaccard's coefficient, ranged from 0.92 to 1.0. On the basis of the polymorphisms detected, the analysed strains can explicitly be grouped into two major clusters, equivalent to two evolutionary lines of the organism found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes.     

土壤细菌16SrRNA基因变异型及其与植被的相关研究

绕过细菌的分离培养,直接提取土壤DNA,扩谱,克隆土壤细菌群体的16S核糖体RNA基因（16S,rDNA),根据该基因各种变异类型的限制性片段长度多型性,分析土壤细菌分子遗传多样性及其与植被的相关关系,植被的改变影响土壤养分,进而改变土壤细菌群落结构,土壤细菌遗传多样性和分化能反映植被的变化。     

Molecular cloning and enzymatic expression of the 28-kDa glutathione S-transferase of Schistosoma japonicum: evidence for sequence variation but lack of consistent vaccine efficacy in the murine host

Glutathione S-transferases (GSTs) have long been regarded as attractive vaccine (and drug) targets in schistosomes due to their suspected role in detoxification processes. Indeed, the 28-kDa GST of Schistosoma mansoni (SmGST28) has proven efficacy as an antigen for protective immunity reducing worm burden, female fecundity and egg viability. In contrast, the vaccinating effects of the bacterial expressed homologue of Philippine S. japonicum (SjpGST28) have proved disappointing, possibly because this recombinant form was an incomplete sequence, lacking five N-terminal amino acids which may have affected its vaccination efficacy. Here we describe the cloning and functional enzymatic expression of a complete cDNA encoding SjpGST28. We report also on the immunogenicity and vaccine efficacy of this molecule as a purified recombinant protein and as a DNA plasmid vaccine in the murine model. We further describe the cloning of several complete cDNAs encoding the Chinese homologue of SjpGST28 and the identification of 3 SjcGST28 sequence variants which are probably encoded by distinct alleles.     

植物抗病的分子生物学基础

随着分子生物学的不断发展,人们已逐步了解植物寄主与病原之间的相互作用及植物抗病的分子机理。植物受病原侵染后出现两种类型的卫反应：局部防卫反应（过敏反应）和系统获得性防卫反应。本质素、植保素、活性氧、水杨酸等物质已被证明了在植物抗病中起了重要作用。抗病基因和防卫基因的诱导表达构成了防卫反应的遗传基础。本文综述了近年来抗病的分子生物学研究进展,并对其发展和应用前景作了展望。