Dissertation > Excellent graduate degree dissertation topics show

Expression, Characterization, and Mutant Construction of Oligopeptide Permease from Vibrio Harveyi

Author: HeQingFang
Tutor: ChenJiXiang
School: Ocean University of China
Course: Microbiology
Keywords: Vibrio harveyi oligopeptide permease expression characterization phospholipase C hemolytic activity mutation pathogenicity
CLC: S941.4
Type: Master's thesis
Year: 2011
Downloads: 12
Quote: 0
Read: Download Dissertation

Abstract


Vibrio harveyi is a Gram-negative bacterium, which widely existed in marine environments and marine animals such as fish and crustaceans. The bacterium has been reported as major causal agent of luminous vibriosis, which affected a diverse range of marine invertebrates. Mass mortalities of shrimp larvae due to V. harveyi infection have been reported from a number of countries. V. harveyi has also been associated with fish diseases in China and South Asian countries.Some Gram-negative bacteria such as E.coli have three peptide uptake systems with varying substrate specificities, allowing them to utilize a broader range of peptides for nutrition purpose: dipeptide permease (Dpp), tripeptide permease (Tpp) and oligopeptide permease (Opp), which handles peptides of two to five residues. Opp is ATP-binding cassette transporter, and composed of five subunits: two transmembrane protein (OppB, OppC), two ATP binding proteins (OppD, OppE) and an oligopeptide binding protein (oligopeptide permease A, OppA). Studies have revealed that bacterial oppA is a solute-binding protein (SBP) and plays an important role in transport of oligopeptides into the cell and in various signaling processes. Increasing evidence indicated that the ABC type transporter proteins take part in the bacterial pathogenic processes.An oligopeptide permease A (oppA) gene was cloned from the genome of V. harveyi SF-1.A. Sequence analysis showed that the cloned gene consisted of 1665 base pairs and encoded a 554 amino acid protein. It showed high similarity to the OppA of V.vulnificus and the ABC-type oligopeptide transporter sequences from V.parahaemolyticus and V.fluvialis. The gene was subcloned into pET-28a (+) and expressed in Escherichia coli. The expressed recombinant protein was purified by Ni-NTA metal affinity chromatography and showed a 58 kDa band on SDS-PAGE. The protein exhibited phospholipase C activity. The optimum temperature and pH was 55 degree centigrade and 8.0, respectively. It was stable at pH 6-9 and below temperature of 55 degree centigrade. In addition, the recombinant protein exhibited lecithin activity and hemolytic activity on the TSB plates adding lecithin and LB plates coated with sheep erythrocytes. A oligopeptide permease mutant of V. harveyi SF-1 was constructed by homologous recombination. Virulence of the oppA mutant strain was also decreased when they were intraperitoneally inoculated into zebra fish, the LD50 of the wild-type and the mutant were 3.11×104 and 5.46×105 CFU per fish respectively. These results indicated that the oppA of V. harveyi played important roles in bacterial pathogenicity.

Related Dissertations

  1. Research on Combinatorial Regulation of Multiple Transcription Factors,Q78
  2. The Research of the High-Rise Building’s Form and Expression in the New Century,TU971
  3. Research on Design of Teaching Buildings for Arts Departments of Higher Learning Institutions,TU244.3
  4. The convergence of mathematics teaching in primary and secondary schools under the new curriculum with the problem representation,G633.6
  5. Research on Temporal Information Recognition and Normalization,TP391.1
  6. Expression of D-AtCGS in E. Coli and Preparation of Polyclonal Antibody Against D-AtCGS,Q943.2
  7. The Study on Arabidopsis Thaliana Heat Shock Factor HSFA 1d Response to Formaldehyde Stress,Q945.78
  8. Expression of hBMP4 and hBMP7 in Chinese Hamster Ovary Cells,Q78
  9. Cloning and Expression Analysis of GPx, GST and SAHH Genes in Chlamydomonas Sp. ICE-L from Antarctica,Q943.2
  10. Study of Mutation and Breeding of Griseofulvin Produccing Strain FS80,TQ927
  11. cDNA Cloning, Expression of vp5 and vp7 Genes and Subcecullar Localization of VP5 and VP7 Proteins in Grass Carp Reovirus,S941.41
  12. Cloning, Expression of vp6 and ns38 Genes and Immunogenicity of VP6 and NS38 in Grass Carp Reovirus,S941.41
  13. Expression of Prolyl Isomerase Pin1 in Osteosarcoma and the Effect of Regulation on Cell Cycle,R738.1
  14. The Expression Survey of BMP Signalling Pathway in the Human Embryonic Tooth Germ,R78
  15. Preliminary Study of Phosphorylation Effect on UGT1A3 Metabolism Activity Towards Drugs,R346
  16. Cloning and Expression of Growth Hormone Gene in Cobia (Rachycentron Canadus),Q786
  17. Studies on Synthesis, Characterization and Properties of Fe-V Co-doped TiO2 Catalyst,O614.411
  18. Cloning and Characterization of BAFF Gene from Whitespotted Catshark and Crucian Crap,S917.4
  19. Characterization of Corn Lectin and Application to Screen of Growth-promoting Bacteria from Corn Rhizosphere,S513
  20. Optimization of Fermentation Conditions, Purification, Cloning and Expression of a Cold-active Lipase from Pseudomonas Sp.RT-1,TQ925
  21. Breeding of Fungal α-amylase High-producing Strain by Genome Shuffling,TQ925

CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fish Diseases > Microbial fish diseases
© 2012 www.DissertationTopic.Net  Mobile