Dissertation > Excellent graduate degree dissertation topics show

Cloning and Expression of Type Ⅲ Secretion System Genes of Aeromonas Hydrophila and Regulation of Quorum Sensing System

Author: WangJinXing
Tutor: LiuYongJie
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Aeromonas hydrophila typeⅢsecretion system ascV aopN Quorum sensing system Real-time quantitative RT-PCR
CLC: S852.61
Type: Master's thesis
Year: 2008
Downloads: 92
Quote: 0
Read: Download Dissertation

Abstract


Aeromonas hydrophila is the causative agent of fatal hemorrhagic septicemia in fish and can cause a wide variety of human infections.The bacterial virulence involves multiple factors,including extracellular toxins,proteases and so on.And TypeⅢsecretion system (TTSS) and quorum sensing(QS) might play an important role in pathogenicity.1.One pair of primers was designed based on the conserved ascⅤgene sequences from TTSS of A.hydrophila published in GenBank.A 331bp DNA fragment was amplified from the genomic DNA of A.hydrophila J-1 strain by PCR.The PCR product was inserted into pMD 18-T and sequenced.Considering that the homologies of ascⅤgenes were very low in the different strains of A.hydrophilas,the whole ascⅤgene fragment was obtained by cloning four fragments and merging.The ascⅤnucleotide sequence of J-1 were 97%,86%, 86%,86%and 87%identical to those of A.hydrophila strains AH1,SSU and AH3,A. salmonicida subsp salmonicida A449 and A.sobria,respectively.Sixty-six strains of A. hydrophila were detected for ascⅤby PCR method,and 64 strains were positive.It suggested that the typeⅢsecretion system is commonly present in the different strains of A. hydrophilas.The PCR results,in which two virulent strains were negative for ascⅤ,while two avirulent strains were positive,indicated that TTSS might not play an important role in the pathogenisis of some virulent strains of Ah.2.One pair of primers with EcoRI and XhoI sites were designed according to the nucleotide sequence of aopN gene from TTSS of A.hydrophila strain AH-1.With the specific primers,the 874bp fragment was obtained from the Ah J-1 genome by PCR.The aopN gene was then sequenced and analyzed.The aopN nucleotide sequence of Ah J-1 was 97%,81%,82%,81%and 82%identical to those of A.hydrophila strains AH1,SSU,AH3, A.salmonicida subsp salmonicida A449 and A.sobria,respectively.Sixty-six strains of A. hydrophila were detected for aopN by PCR method,and 57 strains were positive.After the fragment was digested with EcoRⅠand XhoⅠ,it was cloned into the expression,vector pET-32a(+) digested with EcoRⅠand XhoⅠ.The recombinant pET-aopN vector was transformed into E.coli BL21 and induced to express by IPTG.The result of SDS-PAGE showed one band of 54.5kDa as expected.Moreover,the fusion protein could be recognized by antiserum to Ah J-1 in Western blotting,indicating that that the recombinant AopN has immunoreactivity and maybe the common protective antigen in the different strains of A.hydrophila.3.TTSS and QS play an important role in the pathogenicity of many pathogenic bacteria.To understand their relationship,an ultrasensitive bioassay system was constructed in Agrobacterium tumefaciens by using the T7 expression system to detect autoinducers(AIs) made by A.hydrophila J-1 and mutant strain J-1△ahyR.The result showed the production of AIs was lower in J-1△ahyR.It suggested that ahyR gene is an important regulation gene.Based on the result,real-time quantitative RT-PCR was used to detect the expression level of AopN mRNA in A.hydrophila J-1 and mutant strain J-1△ahyR.Two pairs of primers were designed according to the 16S rRNA gene sequence published in GenBank and aopN gene sequence in the second chapter.The total RNAs were extracted from A.hydrophila J-1 and J-1△ahyR,respectively.The result indicated that the expression level of AopN mRNA in A.hydrophila J-1 was lower than that in the mutant strain J-1△ahyR.It suggested the quorum sensing system might regulate the typeⅢsecretion system negatively in A.hydrophila J-1.This study is the backbone for further investigation on the relationship between TTSS and QS.

Related Dissertations

  1. Studies on the Mechanism and Application of Bacillus Subtilis Lipopeptide Anti-Aeromonas Hydrophila,S948
  2. Cloning of PsMAPK1, PsCdc2, PsFuz7 and Functional Characterization of PsMAPK1 from Puccinia Striiformis F.Sp. Tritici,S435.121
  3. Studies on Anguilla Anguilla Intestine Probiotics for the Prevention and Cure of the Disease,S94
  4. Rana Aeromonas Hydrophila Aerolysin Gene Pronucleus Expression and Preliminary Analysis of Immunogenicity,S947.2
  5. Bacillus subtilis JS01 on Aeromonas hydrophila infection carp digestion and absorption capacity and immune function,S943
  6. Effects of Aeromonas Hydrophila and Environment Factors on Immune-related Indexes of Procambarus Clarkii,S945
  7. Isolation and Identification of Pathogen from Black Carp Enteric Hemorrhage and the Preliminary Study of Vaccines,S943
  8. Preliminary Study on Out Membrane Structure and Immunogenicity Changes of Drug-resistant Aeromonas Hydrophila,S941
  9. The Virulence of the Aeromonas Hydrophila,S941
  10. Studies on Modulating Effects of Bacterial Genomic DNAs on the Immune Function of Chinese Mitten Crab Eriocheir Sinensis,S917.4
  11. Effect of Dietary Supplementation of Probiotics As Immunostimulants on Growth Performance, Immune Responses and Intestinal Flora of Japaness Sea Bass (lateolabrax Japonicus),S963
  12. ELISA was used to detect Aeromonas hydrophila and Flavobacterium,S941
  13. Study on Preparation of Genetic Engineering Live Vector Vaccine of Outer Membrane Protein of Aeromonas Hydrophila and Its Immune Effect in Mice,S852.5
  14. Development of Kits for the Diagnosis Patnogenic Aeromonas Hydrophila,S854.43
  15. Analysis of Types Virulence Genotypes in the Different Strains of Aeromonas Hydrophila and Expression, Extraction and Analysis of Extracellular Protease,S852.61
  16. Aeromonas hydrophila isolated from the outer membrane protein profiles of different analysis and relationship with drug resistance,R115
  17. Fish source Aeromonas hydrophila Isolation, Identification and Drug Resistance,S941
  18. Analysis the Major Adhesin Gene Sequences and It’s Recombinant Gene Products Function of Aeromonas Hydrophila,S941
  19. Studies on the Pathogens of Hemorrhage Disease and Parasitic Helminths in Rice Field Eels(Monopterus Albus) in Sichuan Province, China,S943
  20. Study on Preventive Measures for Aeromonas Hydrophila and Aeromonas Sobria in Rice Field eel (Monopterus Albus),S943
  21. Hydrophila Aeromonas in Siberian sturgeon in vivo distribution of pathological changes,S943

CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
© 2012 www.DissertationTopic.Net  Mobile