Analysis of Aeromonas hydrophila Serine Protease Gene prtS1 and Fabrication for Deficient Strain
| Autor: | Chun-Chung Wang, 王寵鈞 |
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| Rok vydání: | 2002 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 90 Aeromonas hydrophila and related aeromonads are gram negative, facultatively anaerobic fresh water bacteria, many strains of which are pathogens of humans and animals. The pathogenicity may involve with several extracellular enzymes including haemolysins, lipase, and protease. It has been suggested that proteolytic enzymes produced by A. hydrophila may play an important role in invasiveness and in establishment of infection by such as overcoming initial host defense and providing nutrients for cell proliferation. During previous study, we have cloned and sequenced a proteolytic gene prtS1 from clinical strain A. hydrophila CKH29. prtS1 can be translated into a 453 a.a. protein which is similar to HtrA/DegQ/DegS family protease and may act as protective factor response to environmental stresses during infection. In this study we characterized the properties of gene prtS1. We analyse the existence of prtS1 from 20 clinical strains and 30 environmental strains by PCR reaction and Western bloting. Similar fragment of prtS1 can be amplified from genomic DNAof all strains after analyzed by PCR. But we can’t find any fragment amplified from genomic DNA of other bacteria. It suggested that prtS1 is a common and specific gene among A. hydrophila. In Western blotting analysis, the PrtS1 wcab be detected from most strains of A. hydrophila. In the comparison of A. hydrophila type strain CCRC13880 with CKH29 of DNA sequences and protein molecule, the DNA sequences have idensity of 88% and a.a identity of 96.7%. The different amino acids are all located in PDZ domain of PrtS1 C-terminal. It suggested that the differences of molecular weight between A. hydrophila CCRC13880 and CKH29 is related to their PDZ domain. To study on the function of prtS1 in A. hydrophila CKH-29, we have constructed a plasmid pSup2021-PrtSM ( pSup2021::PrtS1::Km ), and tried to let the plasmid integrating into CKH29 chromosome through conjugation. The conjugation frequency is 1×10-5 and we afforded to get isogenic mutant strain by means of homologous recombination replacing prtS1 with prtSM. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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