Study On The Expression Change Of Lysozymes And Its Immune Effect In Grass Carp(ctenopharyngodon Idellus)after Infected With Aeromonas Veronii

In recent years,various fish diseases had occurred frequently due to improper breeding methods and the destruction of the water environment,which has brought great economic losses to the aquaculture industry.Aeromonas veronii could cause grass carp(Ctenopharyngodon idellus)suffer from septicaemia,causing a large number of deaths in the short term.A strong toxic A.veronii AvX005 was screened from the diseased grass carp by the research team in the early stage.Grass carp,as a lower vertebrate,non-specific immunity plays an important role when exogenous microorganisms invade,and lysozyme is a key part of non-specific immunity.This paper mainly studies the mechanism of grass carp g-type lysozyme and c-type lysozyme as immunestimulants to protect grass carp from AvX005 infection,which has certain value for the research and application of grass carp health breeding.In order to explore the expression changes of g-type lysozyme and c-type lysozyme of grass carp after being infected with AvX005,qRT-PCR was used to analyze the expression levels of Lys-g and Lys-c of grass carp infected with AvX005.The results showed that transcription of the genes lys-g and lys-c in the liver and spleen was significantly induced at 3 hours after infected.In the liver and spleen,the transcription level of the gene lys-g reached a peak at 24 hours after infected,which increased about 13 fold and 4.5 fold,and the transcription level of the gene lys-creached a peak at 12 hours after infected,which increased about 7.5 fold and 27 fold,respectively.Therefore,it is speculated that the two lysozyme proteins play an important role in the early defense of grass carp against AvX005 infection.The recombinant proteins rLys-g and rLys-c were successfully expressed in the recombinant expression system of E.coli.After purification,the recombinant proteins were found to have lysozyme activity by using Micrococcus luteus as an indicator strain,and had lysolytic activity against AvX005,Aeromonas hydrophila Ah X040,and Edwardisella tarda.After overexpressed of rLys-g and rLys-c in liver cells L8824 by transfection,and attacked with AvX005,It was found that compared with the survival rate of the control group(46.3 %),the cell L8824 survival rates of the experimental groups of rLys-g and rLys-c were 77.6 % and 68.6 %,respectively.The results show that recombinant proteins rLys-g and rLys-c could protect cells from AvX005.pcDNA3.1-lys-g and pcDNA3.1-lys-c mixed with adjuvant QCDC were injected into grass carp as immunestimulants.Through attacked with AvX005.It was found that pcDNA3.1-lys-g and pcDNA3.1-lys-c could provide protection for grass carp,all the untreated grass carp died,but preprocessed with QCDC,QCDC+empty,QCDC+g-lys and QCDC+c-lys provided protection rates of 50 %,60 %,70 % and 70 % for grass carp,respectively.The above results showed that the tworecombinant plasmids pcDNA3.1-lys-g and pcDNA3.1-lys-c have the potential to provide protection for grass carp infected with AvX005 as immunostimulants.Grass carp injected with plasmid could induce immune response.After grass carp injected with plasmid,the activity of lysozyme in serum was significantly(P <0.05)increased by about 1.7times and 2 times,and the activity of alkaline phosphatase(AKP)was significantly increased(P <0.05))About 3 times and 2.4 times,the activity of acid phosphatase(ACP)did not change significantly(P> 0.05),and the expression levels of related immune factors C3,IgM and IL8 in grass carp kidney increased to varying degrees.In summary,this study indicate that the expression of g-type lysozyme and c-type lysozyme were significantly induced after grass carp infected with AvX005;The recombinant proteins of lysozyme have lytic activity against AvX005 and could protect grass carp liver cell L8824;pcDNA3.1-lys-g and pcDNA3.1-lys-c have the potential to protect grass carp from AvX005 infection as immunostimulants.