Cloning And Expression Of Type Ⅲ Secretion System Genes Of Aeromonas Hydrophila And Regulation Of Quorum Sensing System

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 Typeâ…¢secretion system (TTSS) and quorum sensing(QS) might play an important role in pathogenicity.1.One pair of primers was designed based on the conserved ascâ…¤gene 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 ascâ…¤genes were very low in the different strains of A.hydrophilas,the whole ascâ…¤gene fragment was obtained by cloning four fragments and merging.The ascâ…¤nucleotide 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 ascâ…¤by PCR method,and 64 strains were positive.It suggested that the typeâ…¢secretion system is commonly present in the different strains of A. hydrophilas.The PCR results,in which two virulent strains were negative for ascâ…¤,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 EcoRâ… and Xhoâ… ,it was cloned into the expression,vector pET-32a(+) digested with EcoRâ… and Xhoâ… .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-1â–³ahyR.The result showed the production of AIs was lower in J-1â–³ahyR.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-1â–³ahyR.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-1â–³ahyR,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-1â–³ahyR.It suggested the quorum sensing system might regulate the typeâ…¢secretion system negatively in A.hydrophila J-1.This study is the backbone for further investigation on the relationship between TTSS and QS.