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Expression, Purification and Characterization of the TLH Hemolysin of Vibrio Campbellii

Author: SunZuo
Tutor: ZhangXiaoHua
School: Ocean University of China
Course: Cell Biology
Keywords: Hom Vibrio Hemolysin Expression Protein Purification Hemolytic activity
CLC: S941
Type: Master's thesis
Year: 2010
Downloads: 87
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Abstract

In recent years, with the continuous development of the mariculture industry, aquaculture disease was aggravated trend, one of the primary problem restricting the development of fish farming. The most harmful in the most serious types of aquaculture diseases, bacterial diseases, pathogens among which Vibrio (Vibrio). A wide range of vibrio disease epidemics, spread quickly, and the high morbidity and mortality, and thus become the focus of seawater breeding and research. Hom Vibrio (Vibrio campbellii) is a gram-negative, facultative anaerobic marine bacteria, is an important opportunistic pathogen of mariculture animals, red leg disease can cause outbreaks of farmed shrimp, abalone suffering from sepsis strain associated with the onset of snails and breeding juveniles, causing huge economic losses to the mariculture industry. Hom understanding of pathogenic Vibrio late, its pathogenic mechanism is not deep enough. The hemolysin is one of the most widely distributed pathogenic Vibrio toxins, play an important role in the pathogen Vibrio infection process. Although different pathogens Vibrio hemolysin have hemolytic activity, but their amino acid sequences, hemolytic activity and the mechanism of action is different. Preliminary study found that contains Hom Vibrio the Vibrio harveyi VHH hemolysin gene sequences very similar TLH hemolysin gene, the similarity of the nucleotide sequence of Vibrio harveyi VIB645 vhhA and vhhB respectively 79.81% and 79.73% of its amino acid sequence and VHH hemolytic similar factors of 85%, both hemolysin in the 147-406 amino acid position at a lipase domain (Lipase_GDSL), speculated that this class of hemolysin phospholipase. Cloning in the expression of this study Hom Vibrio the tlh gene, recombinant expression, was purified by Ni-column; analysis of the hemolytic activity of the purified TLH hemolysin and hemolysin by the influence of temperature, pH and metal ions, and further TLH hemolysin pathogenic zebrafish. We designed a pair of specific primers, since Hom Vibrio genome amplified tlh hemolysin gene (1254bp); The PCR products were ligated into the expression vector pET-26b () built Hom Vibrio TLH hemolysin The recombinant expression plasmid pET26b () / tlh, and transformed into E. coli BL21 (DE3); adding a final concentration of 1mM IPTG induced recombinant bacteria expressing TLH with six histidine-tagged fusion protein. After induction of recombinant Escherichia coli and its culture supernatant in the fresh Japanese flounder blood agar 16 ° C overnight and lecithin Tablet 37 ℃ overnight culture, the culture of hemolytic activity and phospholipase activity induced bacterial cells and supernatant were . SDS-PAGE analysis shows induced expression of the hemolysin in the recombinant Escherichia coli can be mass molecular weight of about 42 kDa. Ni agarose affinity chromatography column reorganization hom Vibrio TLH hemolysin purified purified TLH hemolysin as a single protein on SDS-PAGE electrophoresis FIG belt; drawn using the method of Bradford protein standard curve to obtain a purified The TLH hemolysin concentration of 0.41 mg / mL. The hemolysin activity experiments showed that the purified TLH hemolysin has strong hemolytic activity (hemolytic circle diameter of 16 mm) and the phospholipase activity (turbidity inner ring diameter of 11mm, transparent outer ring diameter 15mm). TLH hemolysin hemolytic dynamic optimum temperature of 37 ° C; temperature stability tests showed, compared with less than 37 ℃ stable and incubated at 45 ° C for 30min to lose 50% of its vitality, and incubated for 30 min at 55 ° C for its vitality loss 75% incubated at 75 ° C for 30 min and the complete loss of hemolytic activity, the card Mingkan Vibrio TLH hemolysin thermal instability hemolysin. TLH hemolysin optimum pH of 6, at a pH of 5 and 7 hours, only 50% activity, and TLH hemolysin in pH 5-10 relatively stable and pH greater than 10 or less than 5 will lead to its stability decline rapidly. Affect the outcome of this experiment, 10 kinds of metal ions (Na, K, Mg2, Ca2, Mn2, Co2, Ni2, Cu2, Zn2 and Ba2) Hom Vibrio TLH hemolysin hemolytic activity, a divalent metal ions such as Na K on the hemolytic activity is almost no effect, while the portion divalent metal ions such as Ca2, CO2 would lead to hemolytic activity is decreased, indicating that some of the divalent metal ion can be suppressed hom Vibrio TLH hemolytic activity of hemolysin. The pathogenicity studies show that the TLH hemolysin infect healthy zebrafish, the zebrafish chest and abdominal cavity bleeding swelling and systemic bleeding, and zebrafish lethal effect, the median lethal dose LD50 2.158μg / g fish. The results of this study is important to clarify Hom Vibrio pathogenesis and genetic engineering vaccine development.

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