Construction Of Phage Display Single-chain Fv Antibody Library Against UV-attenuated Schistosoma Japonicum Cercariae And The Screening Of The Sj SCA66-68kDa ScFv

ObjectiveTo construct the highly efficient UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library and to obtain the SCA66-68kDa ScFv based on the advantages of UV-Attenuated Cercariae Vaccines,natural molecular vaccine and phage antibody library technology,inorder to get the new candidate vaccine coding genes of SjSCA66-68,which would advance the progress of vaccine for schistosoma japonicum.Methods1.Using the methods of phage display antibody library to construct the single-chain Fv(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae and characterize it from the way of capacity and diversity.BALB/c mice were immunized several times with UV-attenuated Schistosoma japonicum cercariae and In order to evaluate the protection potential of UV-attenuated cercariae serum, the serum was transferred passively to mice at 1 week before,1 week after and 3 weeks after challenged with Schistosoma japonicum cercariae.Once we get the protective serum,the spleens of these mice was used to prepare RNA.The variable heavy(VH)and variable light(VL)genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR.The scFv fragments were cloned into the vector PCANTAB 5E and the recombinant plasmids were electroporated into competent E.coli TG1 cells.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was obtained after the recombinant phagemids were rescued by infection of helper phage M13K07.2.Confirm the target screening antigens with highly efficient UMS and the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68:SjSCA66-68 were purifed from SjSCA as following procedures:SDS-PAGE,gel band secton, electroelution and ultrafiltraton,etc;The monospecific anti-sera against SCA66-68 was produced through immunization of New Zealand rabbit in the way of injection with microdosis SCA66-68 antigen gel into lymph node;Characterize the SCA66-68 antigen through silver and PAS staining;Sequence the SCA66-68 protein with N-terminal sequence analysis.3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68 through the methods of NC meberance enrichment and colony lift assaydouble binded with the way of antibody sandwich method and antigen direct method respectively.The specificity of SCA66-68kDa ScFv was identified by western blot.Results1.BALB/c mice were transferred passively with UV-attenuated cercariae serum,the results showed the UV-attenuated cercariae serum conferred significant higher level of protection(42.5%)when transferred before challenge than that of natural infection(28.0%).As the base of the high protective animal model which induced by UV-attenuated cercariae,UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library were construct successfully with the capacity of 1.9*10~8 clones and the recombination rate of 100%and good diversity.2.66-68kDa antigen in different stage of schistosoma japonicum could be recognized by highly efficient UMS and SCA66-68kDa was taken as t the target screening antigens;SjSCA66-68 were purifed from SjSCA and the rabbit monospecific anti-sera with a titer as high as 1:12800 against SCA66-68 were produced successfully;The SCA66-68 antigen was characterize as a non-glycoprotein through silver and PAS staining;The result of N-terminal sequence analysis showed the N-terminally blocked proteins of SCA66-68,so we put forward some suggestions on the strategy of the sequencing of SCA66-68kDa.3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68,after enriching and screening,we get six positive clones in the way of antigen direct method,and no positive clones with the antibody sandwich method.The Western blot analysis and SDS-PAGE showed that specific ScFv SCA66-68 was successfully abtained after Soluble expression of E-coli HB2151.Conclusion1.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was successfully constructed.The construction of this library may overcome the limitation of the application of radiate-attenuated vaccine,and benefit to develop the new strategies of prevention on Schistosoma japonicum.2.SjSCA66-68kDa nature antigen could be recognized by UMS,the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68 provides a theoretical basis for further screening of ScFv library,also lay a firm foundation for developing effective molecules of vaccine against schistosoma.3.Successfully get the specific SCA66-68kDa ScFv with the methods of NC meberance enrichment and colony lift binded with antigen direct way,which has shown the wide application prospects in the field of the vaccine,immunodiagnosis and Immunotherapy.