Production And Applications In Cell Sorting And Vaccine Evaluation Of Monoclonal Antibody To Large Yellow Croaker(Larimichthys Crocea)IgM

Large yellow croaker(Larimichthys crocea,lyc)is an economically important fish cultured in China.Lyc's bacterial disease,parasitic disease and virus disease broke out frequently in some aquaculture bases as a result of the enlargement of the cultivation scale in recent years.So far,the prevention of lyc's bacterial disease depends mainly on antibiotics,which may lead to the resistance of pathogen increasing,in the other hand,the abuse of antibiotic may lead to antibiotic residue and we'll be self-inflicted.Vaccination is the main way and development direction of immune control of fish diseases.Therefore,the research on molecular and cytological,and development of vaccine against important pathogenic bacteria will provide a theoretical basis and new approach for the immune prevention and control of the bacterial disease of the large yellow croaker.DLD(dihydrolipoamide dehydrogenase),which is an important molecule in the energy metabolic chain of living organisms.DLD is widely found in living organisms..We cloned DLD from Vibrio alginolyticus,recombinant pet-28a-DLD and purified it by E.coli BL21 expression system.The recombinant DLD was used to inject into large yellow croaker,and found the activity of lysozyme in serum increased significantly.At the same time,the IgM antibody of large yellow croaker showed that the antibody titer of DLD in the serum of the large yellow croaker was always high at 60 d after immunization(up to 1:256).In addition,in the challenge experiments of Vibrio alginolyticus and Vibrio parahaemolyticus,the RPS of DLD immunized with DLD was about 65%,showing the potential of DLD as a subunit vaccine.In order to further understand the cytological basis for the immune response of bacterial vaccine,the IgM+ B lymphocyte of large yellow croaker was isolated and identified.In this research,lycIgM was primary purified by Protein A-Sepharose FF.Large yellow croaker tetramer IgM(about 800 kD)exists in serum.SDS-PAGE analysis showed the H chain was 74 kD,L chain was 25 kD.The Balb/C mice were injected i.p.by lycIgM with buffer,we judged the antibody titer of mice serum and spleens were taken for fusion.Positive hybridomas were cloned 3 times,Mab was produced and purified.The specificity and availability of mAB was detected by Western blot(WB),and immunofluorescence assay(IF);Further,we build up magnetic activated cell sorting(MACS)and sorted positive cells,then B cell molecule marker was detected.This is the first mAB available in lye B cells sorting,and producing condition for further research in large yellow croaker immune system.Above all,we identdified a poteintial vaccine for large yellow croaker vibriosis,produced a monoclonal antibody available in B cell sorting of large yellow croaker successfully and identified B cells.This helped building up the vaccine evaluation system and lay the basis for further study in large yellow croaker immune system.