Characterization And Gene Cloning, Expression Of A Lipase Of Aeromonas Hydrophila J-1

Aeromonas hydrophila is a gram-negative opportunistic pathogen in humans andseveral fish species, causing soft tissue wound infections and diarrhea in the former andfatal hemorrhagic septicemia in the latter. A. hydrophila could secret a repertoire ofexoenzymes which digest cellular components such as proteases, amylases, and lipases.These exoenzymes are involved sequentially as the bacteria colonize, gain entry, establishthemselves, replicate, cause damage in host tissues, evade the host defense system, andspread, eventually killing the host. Therefore, the purpose of the present study was to gainthe complete lipase gene from Ah J-1 by Genome walking technique and establishfoundation for researching the function of lipase further.1. Lipase produced by Aeromonas hydrophila strain J-1 was purified and characterized.Lipase was successfully purified by ultrafiltration through 10kDa nominal molecularweight cut-off membrance, 25%-50% ammonium sulfate fractionation, anion-exchangechromatography and sephaceryl chromatography. SDS-PAGE analysis showed that lipasewas 80kDa protein. The optimum pH and temperature of purified elastase was pH 7.4 and37℃, respectively. The lipase did not caused death of mice by intraperitoneal injection andcytopathic effect (CPE) of HEp-2 cell by co-culture.2. A 383bp gene fragment within the conserved region of lipase gene from A.hydrophila J-1 was obtained by PCR using primers reported. Subsequently, Genomewalking technique was applied to amplify unknown DNA sequences flanking the 383bpconserved fragment. Genome walking libraries were constructed using six restrictionenzymes (DraⅠ, EcoRⅤ, PvuⅡ, ScaⅠ, StuⅠ, SmaⅠ) and then used as templates to amplifythe unknown flanking sequences. By linking the flanking sequences with the 383bpfragment, we got a long fragment up to 3476bp. Sequencing analysis revealed a 2415bpopen reading frame. Furthermore, BLAST analysis shows the 2415bp ORF has 97%, 97%, 88%, 83% and 82% homologs to the DNA sequences of A. hydrophila ATCC 7966 lipase,A. hydrophila AH-3 phospholipase A1, A. hydrophila H3 lipase, A. hydrophila Mcc-2lipase and A. hydrophila JMP636 phospholipase C, respectively. The deduced 805 aminoacid sequence contains a sequence of VHFLGHSLGA which is very well conserved amonglipases. We detected the distribution of lipases in 35 Aeromonas isolates by PCR andphenotypic test using tributyrin as substrate. The positive rates were 100% and 83%,respectively. The lipase activity or associated genes could be detected in the two avirulentstrains, A. hydrophila W1 and A. hydrophila 4332. It suggested that lipase might not be animportant factor in pathogenic mechanism of A. hydrophila.3. A pair of primers was designed based on the nucleotide sequence acquired bygenome walking. With the specific primers, the complete lipase gene was gained by PCRfrom the Ah J-1 genome. The internal fragment was recovered by EcoR I and Xho Irestriction digestion and finally ligated into the EcoR I and Xho I digested expressionplasmid vector pET-32a(+). The recombinant pET-Lip vector was transformed into E.coliBL21 and induced to express by 1mmol/L IPTG at 37℃. An expected 80kDa fusion proteinwas expressed. SDS-PAGE analysis showed the fusion protein located in the supernatant ofbacteria lysate by sonication. The fusion protein was purified by HisTrap^TM HP Kit and coulddegrade tributyrin. The lipase did not caused death of mice by intraperitoneal injection andcytopathic effect (CPE) of HEp-2 cell by co-culture.