镜鲤骨骼肌酵母双杂交cDNA文库的构建及鉴定

为了研究蛋白质之间的相互作用,利用Gateway技术构建了镜鲤(Cyprinus carpio Linnaeus)骨骼肌酵母双杂交cDNA文库.经检测表明,构建的初级cDNA文库的库容量为8×106CFU;酵母双杂交cDNA文库的库容量为6.64×106CFU,插入片段集中在1-2 kb之间,具有较好的多态性.随机挑取的24个克隆没有空载体,全部发生了重组.较高的库容量、较长的插入片段以及较高的重组率保证了文库的完整性和覆盖度.镜鲤骨骼肌酵母双杂交cDNA文库的构建为克隆全长目的基因及研究影响骨骼肌发育的信号传导通路奠定了基础.     

与胡萝卜胚胎发生相关的胚性细胞蛋白63cDNA分离及其基因表达研究

以胡萝卜(Daucus carota L.)鱼雷形胚状体为材料,以λgt10噬菌体为载体,构建了一个含有6.0×10~8个重组子的cDNA文库。用PCR法扩增的长度为1.1kb的胚性细胞蛋白(ECP)63 DNA片段作探针,从cDNA文库中筛选出一个完整的ECP63 cDNA克隆。ECP63 cDNA核苷酸序列总长为1989bp,编码1个含569个氨基酸残基的蛋白质,分子量为62kD。以ECP63 cDNA全长作探针的Northern分子杂交结果表明,ECP63基因在胚性细胞和不同发育时期的胚状体中高度表达,但在幼苗和非胚性细胞中不表达。在转录水平上,ECP63基因在合子胚胎发生后期大量表达。     

SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。     

甜菜夜蛾触角全长cDNA文库的构建与分析

目的:构建甜菜夜蛾触角全长cDNA文库。方法:利用TRIzol试剂提取甜菜夜蛾触角总RNA,以此为模板,通过SMAR-TScribeTM反转录酶反转录合成第一链cDNA,引物扩增获得双链cDNA,经Proteinase K消化、SfiⅠ酶切和CHROMA SPIN-400Column分级分离后,收集400～2 000 bp之间的片段重组于改造的pUC19载体并转化至大肠杆菌Escherichia coli DH5α,最终构建获得甜菜夜蛾触角全长cDNA文库。结果:对文库进行滴度测定和重组率分析,结果表明构建的cDNA初级文库滴度为1.6×107pfu/ml,重组率为94%,插入片段大小为0.5～3.0 kb,平均长度在1 kb以上,表明构建获得的文库是一个高质量的文库。结论:该文库的构建为今后克隆甜菜夜蛾嗅觉相关基因奠定了基础。     

利用改进的Oligo-capping法构建手掌参全长cDNA文库

Oligo-capping法是构建全长cDNA文库的重要方法之一.以名贵中药手掌参(Gymnadenia conopseaR.Br.)的幼芽组织为材料,通过减少mRNA的用量,用pUC18作载体,设计特异引物对第1链cDNA进行PCR扩增,按片段大小分级回收cDNA,形成了一种以Oligo-capping法为基础,构建珍稀材料全长cDNA文库的快速、简单的技术体系.利用该方法,首次成功地构建了手掌参的全长cDNA文库.经综合评价,文库容量达到了8×105个/μg cDNA,全长cDNA比例达到68%,重组率超过96%,说明本实验的改进非常成功.该文库的建成为手掌参的遗传资源保护和功能基因发掘等理论与应用研究奠定了基础.     

盐碱地星星草根cDNA文库的构建及初步分析

以盐碱地原位取材的星星草根为材料,利用SMART技术构建了cDNA文库。扩增后文库的滴度为1.747×109CFU/mL,插入片段分布在0.5-2 kb之间,重组率为92%。文库ESTs序列的初步分析表明,从盐碱地星星草根的cDNA文库中筛选到耐盐相关基因S-腺苷甲硫氨酸合成酶2基因和钙牵蛋白基因的EST片段。该文库可用于进一步从中筛选星星草耐盐基因。盐碱地星星草根cDNA文库的构建为揭示星星草耐盐分子机制、挖掘星星草耐盐基因、培养耐盐植物奠定了重要基础。     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

胃癌823细胞cDNA文库的建立

随着DNA重组技术的发展,某些与细胞癌变有关的基因的分离与克隆越来越受到人们的重视。要达到分离和纯化癌基因及其产物的目的,一个较为有效的方法就是构建cDNA文库。最近我们建立了胃癌823细胞(BGC-823)的cDNA文库,希望能从文库中分离到与胃癌     

草鱼细胞系CIK酵母双杂交cDNA文库的构建及初步应用

旨在探索草鱼呼肠孤病毒与宿主细胞蛋白质间的相互作用,运用SMART技术构建了草鱼肾组织细胞系CIK(Ctenopharyngodon idellus kidney)的酵母双杂交cDNA文库。提取CIK细胞总RNA后,分离纯化mRNA,然后以mRNA为模板,反转录合成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR,扩增双链cDNA。利用SMART技术,通过同源重组的方法,在酵母株Y187中构建了草鱼CIK细胞全长cDNA文库。经检测,未扩增文库的转化率为1.6×105,文库容量为2.4×106,插入的双链cDNA片段的长度为250-2 000 bp,文库滴度为7×107 CFU/mL,重组率为98%,此文库具有良好的cDNA片段多态性和完整性。利用构建的CIK酵母双杂交文库,以草鱼呼肠孤病毒的VP7和VP5蛋白作为诱饵进行筛选试验,得到VP7相互作用蛋白的阳性菌落,未得到VP5相互作用蛋白的阳性菌落。草鱼CIK细胞酵母双杂交cDNA文库的构建为研究草鱼呼肠孤病毒与宿主细胞间的互作机制提供了重要研究工具。     

淫羊藿花蕾cDNA文库的构建与鉴定

基于sm art技术构建了淫羊藿花蕾cDNA文库并检测了其质量。结果表明,该文库重组率为95%,平均插入片段大小为1095 bp,文库滴度为2×106pfu/mL,是一个高质量的淫羊藿花蕾cDNA文库。此文库的建立将有助于克隆与次生代谢相关的基因,特别是淫羊藿黄酮特异合成代谢的基因,其次是克隆与花发育相关的基因。     

O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep

Abstract We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev.l vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.     

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.     

Genome Sequence of Brucella melitensis S66, an Isolate of Sequence Type 8, Prevalent in China

Brucella melitensis is the most-represented Brucella species causing human brucellosis in China. Here we report the complete genome sequence of B. melitensis strain S66, a representative strain of sequence type 8 (ST8), which is prevalent in China, making it possible to compare the genome sequences of isolates from different countries.     

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.     

Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.     

羊布鲁氏菌非编码小RNA分子BSR-2的预测与鉴定

利用RT-PCR和RACE等方法对生物信息学预测的羊布鲁氏菌非编码小RNA(smallnon-codingRNA,sRNA)BSR-2进行了实验鉴定,并通过分析BSR-2在胞内生存缺陷株中的转录情况以及预测BSR-2的靶标基因对BSR-2的功能进行初步探讨.RT-PCR结果表明,BSR-2在羊布鲁氏菌的总RNA中存在转录本,而且在不同的应激条件下转录水平不同.RACE结果表明,BSR-2长224nt,位于Ⅱ号染色体的BMEII0742和BMEII0743之间的基因间区.进一步的实验结果表明,BSR-2可能与布鲁氏菌的胞内生存能力相关.     

布鲁氏菌ery操纵子突变株生物学特性分析

布鲁氏菌ery操纵子参与赤藓醇代谢. 赤藓醇能够促进布鲁氏菌的生长.为进一步研究布鲁氏菌引发宿主流产的分子机制,采用基因重组技术构建布鲁氏菌ery操纵子启动子缺失株（△ery）,通过体内外实验探讨布鲁氏菌ery操纵子的生物学功能. 研究结果显示,获得了布鲁氏菌ery操纵子缺失株;布鲁氏菌ery操纵子缺失株侵染胚 胎滋养层细胞脱落较亲本株明显下降;巨噬细胞CFU计数缺失株作用组和亲本株作用组差异显著（P<0.05）.试管凝集和虎红平板实验结果显示均出现凝集现象;检测血清中细胞因子IL-10和TNF-α的表达水平,△ery诱导机体产生的IL-10和TNF-α明显低于亲本株（P<0.05）.小鼠脾脏细菌CFU计数结果显示,△ery较亲本株毒力明显下降.本研究表明,布鲁氏菌ery操纵子启动子缺失株毒力较亲本株明显下降,为进 一步揭示布鲁氏菌引起流产的致病机制提供了一定的理论依据.     

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.     

Parallel gene loss and acquisition among strains of different Brucella species and biovars

The genus Brucella is divided into six species; of these, B. melitensis and B. abortus are pathogenic to humans, and B. ovis and B. neotomae are nonpathogenic to humans. The definition of gene loss and acquisition is essential for understanding Brucella's ecology, evolutionary history, and host relationships. A DNA microarray containing unique genes of B. melitensis Type strain 16MT and B. abortus 9-941 was constructed and used to determine the gene contents of the representative strains of Brucella. Phylogenetic relationships were inferred from sequences of housekeeping genes. Gene loss and acquisition of different Brucella species were inferred. A total of 214 genes were found to be differentially distributed, and 173 of them were clustered into 15 genomic islands (GIs). Evidence of horizontal gene transfer was observed for 10 GIs. Phylogenetic analysis indicated that the 19 strains formed five clades, and some of the GIs had been lost or acquired independently among the different lineages. The derivation of Brucella lineages is concomitant with the parallel loss or acquisition of GIs, indicating a complex interaction between various Brucella species and hosts.