Detection Of Pathogenic Areomomas Hydrophila And Cloning And Expression And Antigenicity Evaluation Of The Fragment Of Gene Encoding An Extracellular Temperature-labile Protease From Aeromonas Hydrophila J-1

Based on the diagnosis kit for pathogenic Aeromonas, the ECPase54 produced by Aeromonas hydrophila (Ah) was detected by Dot-ELISA, at the same time the gene for specific 16S rDNA and aerolysin of pathogenic Aeromonas hydrophila (PAh) were detected. Ah can be clearly discriminated from other Aeromonas species by 16S rDNA PCR.72 isolates were tested by Dot-ELISA, skim milk plate and PCR method respectively. The agreement between Dot-ELISA and skim milk plate, Dot-ELISA and aer gene PCR, Dot-ELISA and 16S rDNA gene PCR were 79.2%(57/72), 91.6%(66/72), 81.9%(59/72) respectively. The Dot-ELISA experiments were repeated 3 times and the results were correspondent. In conclusion, this Dot-ELISA method is sensitive, specific and practical. It can be used as a method for rapid diagnosis of PAh clinically.With the specific primers, one target fragment about 1005bp which encode an extracellular temperature-labile protease (EprCAI) was amplified from Ah J-l strain genomic DNA by PCR. The PCR products were inserted into pMD18-T vector with conventional protocol and the cloning vector pMD-eprCAI was constructed .The eprCAI was then sequenced and analyzed. The nucleotide sequence of Ah J-l is 91% identical to that of Ah AHl.The nucleotide sequence was predicted to encode a 305aa protein from the 90th nucleotide with the molecular mass 33kDa and with high antigenicity. A PCR assay for rapid and sensitive detection of eprCAI of Ah was developed. The primers based on extracellular protease gene eprCAI of Ah J-l could extend a 387bp fragment. The detection limit for eprCAI by PCR amplification was 1.5×10^-3g/L template DNA. The PCR method is a highly sensitive fast diagnostic tool for the detection of eprCAI from Ah. The results for detection of 21 clinical isolates of Ah suggest that the eprCAI commonly present in Ah.A pair of primers were designed according to the nucleotide sequence of the eprCAI of Ah J-1 strain. With the specific primer, the 850bp fragment was gained by PCR from the Ah J-1 genome. After digesting the fragment with EcoRI+SalI, it was cloned into the expression plasmid vector pET-32a(+) digested with EcoRl+Sall. The recombinant pEY-eprCAI vector was transformed into the expressing strain BL21 and induced expression by lmmol/1 IPTG at 37°C and 30°C respectively. An expected 53.2kDa protein was expressed. The proteolytic activity of the expressed product could be detected, but it could be inactived when treated at 56℃ for 30 min. The recombinant protein was detected by Western blot with Ah J-1 ECPs antiserum, but not with Ah J-l ECPase54 antiserum. The results revealed that in vitro expression of the fragment of eprCAI had the critical antigenitic epitopes of Ah J-l ECPs. The mice were immunized with the recombinant protein. Challenged with germ free culture supernatant of Ah J-l and J-l strain (50LD50) respectively, the relative percentage survival (RPS) to the immunized mice was 60%.