Construction Of Aeromonas Veronii Bacterial Ghost And Evaluation Of Immune Efficacy

Bacterial ghost is not containing bacteria contents (including cell cytoplasm, ribosome,cytoplasmic particles etc.), but its outer cell membrane remained unchanged, which caneffectively identify the exogenous antigen, and can not reproduce dead bacterial morphology.The genetic engineering vaccine generally need adjuvant can play the effectiveness and goodimmunity, but the current knowledge of adjuvant all have toxicity, adjuvant without toxicityand side effects did not appear at present. However, bacterial ghost vaccine itself has adjuvantproperties, with high efficiency immune and good security, so the bacterial ghost is a newvaccine with broad prospect.Aeromonas veronii belonging to the genus Aeromonas, mainly distributed in freshwaterenvironments, the world wide distribution and it main infection the freshwater fish whichimmunity relatively low, bring huge economic losses to the aquaculture industry. At present, forthe prevention and control of the Aeromonas veronii is mainly used antibiotic for fish diseases.But it caused serious environmental pollution, lead to continuous resistant pathogenic bacteria,and the drug residues be harmful to human health. In order to better prevention and treatment ofthe diseases caused by bacteria, this research constructs a new kind of genetic engineeringvaccine (Aeromonas veronii bacterial ghost vaccine), application of fancy carp as experimentalanimal for testing and evaluation of its immunogenicity.The standard experimental application of Aeromonas veronii strains of NC4122-3as theobject of study, analysis and research on its biological characteristics. The test results show thatAeromonas veronii standard strain NC4122-3was gram negative, bacteria are slightly curved,at28~42℃can be growth, but the temperature of28℃multiply more rapidly. Drug sensitivitytest results show that, Aeromonas veronii not sensitive to ampicillin but high sensitivity forgentamicin. Grass carp LD50tests showed that bacterial104.534CFU/mL leads to half the numberof grass carp death.According to the lysis gene E ORF (open reading frame) sequence, specific primers weredesigned including EcoR I and Sal I restriction site. Application of PCR method to amplifygene E, connected to a temperature induced by plasmid PBV220which containing the samerestriction sites of EcoR I and Sal I, the constructed temperature control cleavage of plasmidPBV220-PHE into the gene engineering strain DH5α competent, evaluate the effect of dissolving bacteria on it, cultivation of DH5α28℃(PBV220-PHE) until the OD600value ataround0.3~0.5, at42℃temperature induced,4h tends to be stable, after calculation can knowthe cleavage efficiency can reach99.999±0.001%.Design contains the enzyme locus of BamH I and Xho I specific primers, amplifiedPBV220-PHE promoter, terminator, lysis gene E by PCR method, to obtain2100bp fragment,connect it to the plasmid of PBBR-5-MCS (gentamicin resistance) which have the samerestriction sites of BamH I and Xho I, construction of plasmid MCS-PBV-E, to convert it intothe Aeromonas veronii competent cells were induced by temperature control. The resultsshowed that,42℃cracking6h basic to the lowest value, through the calculation of crackingefficiency which can reach99.9995±0.0005%. The living bacteria detection confirmed that theobtained Aeromonas veronii bacterial ghost vaccine after freezed rying processing have noliving bacteria. By using scanning electron microscope can know, dissolving bacterial channelsgenerally appear in the cell ends and a central location, the bacterial outer membranemorphology remained intact, small fraction of bacterial shrinkage.Use Aeromonas veronii NC4122-3(MCS-PBV-E) bacterial ghost vaccine, formaldehydeinactivated vaccine, PBS respectively immune fancy carp, two weeks after the attackexperiment, evaluate its effect on immuneprotection. The animal experimental results show thatAeromonas veronii NC4122-3(MCS-PBV-E) bacterial ghost vaccine immune protectiveefficiency can reaches as high as80%, but the protection of formaldehyde inactivated vaccineand PBS only can reach66.67%and10%.Based on the Aeromonas veronii NC4122-3(MCS-PBV-E) for temperature inducedregulation, the first building Aeromonas veronii bacterial ghost vaccine, and the vaccinethrough animal experiments show that it is immune to fancy carp can produce high efficiencyprotective. It is expected to become a new and excellent novel vaccine which can preventAeromonas veronii disease in fish.