ABSTRACT
Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonoses. Pathogenesis of the B. abortus infection is complicated, and several researchers have attempted to elucidate the infection mechanism of B. abortus. While several proteins have been revealed as pathogenic factors by previous researchers, the underlying mechanism of B. abortus infection is unresolved. In this study, we identified proteins showing different expression levels in B. abortus mutants with different biological characteristics that were generated by random insertion of a transposon. Five mutants were selected based on biological characteristics, in particular, their growth features. Total proteins of mutant and wild-type B. abortus were purified and subjected to two-dimensional gel electrophoresis. Thirty protein spots of each mutant with expression increases or decreases were selected; those with a change of more than 2-fold were compared with the wild-type. Selected spots underwent liquid chromatography tandem mass spectrometry for peptide analysis. DnaK and ClpB, involved in protein aggregation, increased. SecA and GAPDH, associated with energy metabolism, decreased in some mutants with a growth rate slower than that of the wild-type. Mutants with slower growth showed a decrease in energy metabolism-related proteins, while mutants with faster growth showed an increase in pathogenicity-related proteins.
Subject(s)
Brucella abortus , Brucella , Brucellosis , Chromatography, Liquid , Electrophoresis, Gel, Two-Dimensional , Energy Metabolism , Population Characteristics , Sequence Analysis, Protein , Tandem Mass Spectrometry , ZoonosesABSTRACT
Objective@#The aim was to investigate the genotype distribution of two major epitopes of large surface protein (PreS1) of hepatitis B in Chinese patients and to explore the association between the genotypes of these two epitopes, and to determine whether PreS1 full-length genotype could be revealed according to the polypeptide sequence of key epitopes. @*Methods@#HBV DNA was extracted from the serum of patients for PCR amplification. 278 samples amplified successfully were sequenced and compared with the known HBV sequences in Genbank to determine the two key epitopes of HBV PreS1 genotype (amino acid epitope 21-47 and 94-117, abbreviated as P21 and P94) and PreS1 full-length genotypes. The correlation among three genotyping approaches was analyzed by Cohen’s kappa coefficient to verify the consistency between the key-epitope genotyping and the full-length preS1 genotyping. @*Results@#232 samples were successfully sequenced. The genotyping based on the kind of P21 epitope protein sequence, 201 cases for genotype C, 23 cases for genotype B and 8 cases for uncertain genotypes and genotyping based on the form of P94 epitope protein sequence, 199 cases for genotype C, 25 cases for genotype B and 8 cases for indeterminate genotypes. Lastly, the genotyping based on sequence of the full-length PreS1 sequence, 207 and 25 cases for genotype C and B. P21 or P94 epitope genotyping and PreS1 full length genotyping were highly consistent, respectively, 96.55% and 96.12%, and the two epitopes (P21and P94) genotyping have parallel consistency (93.10%). @*Conclusion@#In this study, an innovatively genotyping method based on the amino acid sequence of key epitopes was proposed. The genotypes of HBV in china were mainly B and C genotypes, and the genotypes of key conserved epitopes of HBV PreS1 were highly consistent with the full-length genotyping ( > 96%). Moreover, genotyping with one or two key epitopes can be used in place of the full-length genotyping.
ABSTRACT
Adaboost algorithm with improved K-nearest neighbor classifiers is proposed to predict protein subcellular locations. Improved K-nearest neighbor classifier uses three sequence feature vectors including amino acid composition, dipeptide and pseudo amino acid composition of protein sequence. K-nearest neighbor uses Blast in classification stage. The overall success rates by the jackknife test on two data sets of CH317 and Gram1253 are 92.4% and 93.1%. Adaboost algorithm with the novel K-nearest neighbor improved by Blast is an effective method for predicting subcellular locations of proteins.
ABSTRACT
Objective: To identify WRKY genes from the rhizomes of Dioscorea zingiberensis and analyze the protein characteristics and expression level of these genes. Methods: The transcriptional EST database of the rhizomes of D. zingiberensis was used to search the analogs of AtWRKY genes by BLASTn, the full-length open reading frames (ORF) of DzWRKY and its protein characteristics were studied using bioinformatic method, and the expression levels of DzWRKY genes in rhizomes and leaves were detected from transcriptional data of the rhizomes of D. zingiberensis. Results: Twenty-seven DzWRKY transcription factors family genes with full length ORF were isolated from the rhizomes of D. zingiberensis, and two WRKY domains were confirmed in six WRKY genes. All the DzWRKY proteins were predicted as hydrophilic proteins and nucleoproteins with highly conserved WRKY domains. The 27 DzWRKY and 19 AtWRKY proteins were divided into three groups by phylogenetic analysis. All DzWRKY genes showed higher expression level in the leaves compared to the rhizomes, and highly expressed genes were mainly in groups I and IId. DzWRKY proteins exhibited lower sequence identity. Conclusion: The DzWRKY genes are successfully isolated for the first time, which would provide a reference for the study on the roles of WRKY in development and active components biosynthesis of the rhizomes of D. zingiberensis.
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Objective To establish a method based on repetitive protein sequences and linear B cell epitope to predict and screen specific peptides of Plasmodium vivax. Methods A P. vivax protein sequence database was reconstructed based on Plas-moDB data,and a customized software for searching of repetitive sequences was used to count the repetition times of each 16 aa peptide in the whole database,and the highly repetitive peptides were chosen to predict the potential linear B cell epitopes. The re-petitive peptides with P. vivax specificity were selected as candidate antigen peptides to synthesize and to couple with KLH carrier protein for immunizing BALB/c mice. After the immunization,the antibody titers of the immunized mice were detected. Results The repetitive information of 16 aa peptides was analyzed by screening of the total 5 432 peptide sequences in the P. vivax data-base. A total of 22 peptides were identified as candidate peptides from the top 1 000 repetitive peptides by linear B cell epitope pre-diction on the BcePred website. Through clustering analysis and similarity comparison,five potential P. vivax specific peptides were selected,synthesized and then coupled with KLH to immunize the mice. The antibody titers of the immunized mice induced by the 5 peptides were all above 1:9 000. Conclusion The method for predicting and screening of specific peptides of P. vivax based on repetitive protein sequences and linear B cell epitope has been established successfully,and all the 5 peptides obtained by the method can induce the high titer antibody in mice.
ABSTRACT
Alignment-free comparison is a recently developed method for sequence alignment, which has high computational efficiency and suitable to the low identical sequences. Alignment-free comparison was successfully applied in the DNA analysis. However, the accuracy of analysis is not high when it was applied in protein analysis because the complexity of protein is larger than DNA by consisting of 20 types of residues. Thus, residues are clustered into a few groups based on their similarity of physicochemical features. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. Therefore, the accuracy of alignment-free comparison is improved.