Isolation And Identification Of The Pathogenic Bacteria From Turbot And The Cloning, Mutation, Expression Of Hemolysin Gene Of Vibrio Anguillarum M3

One pathogenic strain SMP1 was originally isolated from diseased turbot(Scophthalmus maximus) in an outbreak of hemorrgagic septicemia disease at a fishfarm in Qingdao. Sensitivity test showed that norfloxacin ,chloramphenicol, tetracyclinosulfamethoxazole were effective to inhibit the growth of the pathogenic bacterium. Theintramuscular injection experiment revealed the strain was virulent to turbot with an LD50value of 1.94×106 colony forming units/fish. BIOLOG system could not identify thepathogenic strain. The phenotypic feature and biochemical feature of SMP1 was similar tothat of V. anguillarum. The comparative 16S rDNA sequence analyses showed that strainSMP1 exhibited the highest level of similarity to V. anguillarum. The conserved region ofhemolysin gene of V. anguillarum could be amplified by PCR using SMP1 genomic DNAas template. Consequently SMP1 was identified as V. anguillarum. Hemolysin is an important virulence factor in many species of genus Vibrio, but theprevious study on hemolysin from V. anguillarum was not adequately. Using the knownsequence of hemolysin gene of V. anguillarum PT84057, the hemolysin gene of V.anguillarum M3 could not be amplified. Based on the sequence of conserved region ofhemolysin gene of V. anguillarum M3, 5'UTR and part of the hemolysin gene was clonedwith inverse PCR and nested PCR. A genomic library was made by linked with adaptorsafter genomic DNA digested with EcoRV. Then the sequence of the other part ofhemolysin gene was obtained. By comparing the two parts, the sequence of the hemolysingene was obtained. The deduced amino acid sequence was 84% identical to that of V.anguillarum PT84057. The number of amino acids of hemolysin from V. anguillarum M3was 11 less than that from V. anguillarum PT84057. To mutate the hemolysin gene, the conserved region was inserted into suicideplasmid pNQ705, and a chromosomal hemolysin mutant was made via the integration offoreign DNA into the hemolysin gene. The mutated strain was screened by antibiotic andverified by the sequencing of PCR product. The change of virulence was investigated by 3中科院研究生论文 大菱鲆致病菌的分离鉴定和溶血素基因的克隆,突变和表达injecting the mutant and parent strain into fish respectively; compounds of extracellularproducts of the mutant were analyzed by SDS-PAGE. But no significant change was found.Our data suggested that the hemolysin from V. anguillarum M3 might not contribute to thevirulence of the strain. The hemolysin gene of V. anguillarum M3 was then cloned into expression plasmidpET24a and transferred into E.coli BL21(DE3). The hemolysin gene was mainlyexpressed as inclusion body, with a small part of soluble protein. To improve the solublepart, optimization work was done. The result suggested that when the final concentrationof IPTG was 0.1mM, the induction time was 13hr and the temperature was 28℃, theamount of intracellular soluble protein was higher. The molecular weight of the expressedprotein was 8.2 kDa by SDS-PAGE analysis. The induced E.coli BL21(DE3) carryingrecombinant expression plasmid was sonicated ,then the supernatant was incubated withred blood cells of crucian.50mins later, red blood cells were hemolysised. 6×his-tag wasused to purify the expressed hemolysin by binding to NTA-Ni. Under nature condition ,theprotein could not bind to the resin. But under denatured condition, the protein was purifiedsuccessfully. In this study, the second animo acid of hemolysin was changed to Gln from Leu ,but there was no difference between the stability of the two kinds of expressed hemolysin.