Studies On The Antibody Fragment And Immunogenicity Of Outer Membrane Proteins Ofaeromonas Hydrophila Isolate From Eel

To prepare a genetic engineering vaccine and to study its immune protection of eels,we cloned and expressed OMP in Aeromonas hydrophila that is a common pathogen in eels by molecular biological method.At the same time,the specific single-chain antibody fragment(Scfv)is selected from eels phage antibody library with the recombinant expression OMP.We obtained its coding DNA sequence and it was highly expressed in E.coli and yeast.Then biological activities of the expression product was detected and analyzed.This research will lay the foundation for the immunological disease prevention of eels.The main findings are as follows:(1)With full-length gene of outer membrane proteins in Aeromonas hydrophila(B11)as template,we obtained the recombinant expression vector pGEX-4T-2-OMP which was transformed into Ecoli BL21 after cloning,connecting and sequencing.The target protein with the molecular weight about 45 KD induced by 1mM IPTG was obtained.Recombinant OMP dissolved by was used to immunize European eel after purifying and lyophilizing.at the same time there is a heavy moisture inactivated bacterium group and PBS control group.Evaluate the immunity of the body by detecting 14 d,21 d,28 d and 42 d of European eel different tissues about lysozyme activity,serum antibody levels and lymphocyte levels.At the same time in the 28 day perform various eel pathogen attack drug test,study of recombinant OMP immune protection.The results show that: OMP group and Aeromonas hydrophila inactivated group could cause non-specific immune responses of fish,lysozyme content peaked at 21 d or 28 d;OMP group conversion levels in whole blood cells peaks at 21 d,stimulation index significantly higher(P <0.05)than the control group and Aeromonas hydrophila inactivated group;antibody titer of OMP group and Aeromonas hydrophila inactivated group peaked at 28 d,and significantly higher than the control group PBS;attack drug test results showed that the recombinant OMP and Aeromonas hydrophila inactivated vaccine against five different Aeromonas hydrophila strains have different levels of immune protection,recovery rates were higher than PBS control group.(2)The antibody activities of ten antibody plaques selected by the recombinant expression OMP from phage antibody library T7-Scfv,are 68.9%,71.7%,77.7%,77.5%,81.7%,78.4%,77.7%,72.6%,72.8%,68.4%,respectively,in the indirect ELISA experiment.The extraction of genomic phages are analyzed by PCR and sequencing.We named the single chain antibody gene with the highest activity as OMP-Scfv.(3)With OMP-Scfv gene sequence as a template,build prokaryotic expression vector pGEX-4T-2-Scfv and eukaryotic expression pPIC9K-Scfv into e.coli BL21 and yeast GS115,that induced with IPTG and methanol as inducer.E.coli expression product obtained is about 53 KD(where the target protein of about 27 KD,GST-tag of about 26 KD),yeast expression of the protein product of about 27 KD.Expression vector and yeast pPIC9K-Scfv eukaryotic expression in BL21 and yeast GS115,were induced with IPTG and methanol as inducer.For e.coli expression product of about 52 KD(where the target protein of about 27 KD,GST tag of about 26 KD),yeast expression of the protein product of about 27 KD,and detected and analyzed the expression of the biological activity of the product.In summary,the recombinant OMP obtained in this experiment has a strong immunogenicity,for a variety of different Aeromonas hydrophila strains have good immune protection;Through phage antibody libraries T7-Scfv we get the specific single chain antibodies about recombinant OMP,and to achieve expression in E.coli and yeast,and detected and analyzed the expression of the biological activity of the product,for its application in the prevention and treatment of bacterial diseases eel provides a basis.