Identifing The Gene Of Sj31/32 Natural Molecule Vaccine And Screening The Specific Single-chain Antibody

It is proved that the antigen of Sj31/32kDa not only can be used in immunodiagnosis,but also play an important role in anti-fecundity immunity.To further study the antigen of Sj31/32kDa,we analyze it using proteinological methods,we have obtained three highly homology protein spots named number 11,20,23.And they are revelant to Glyceraldehyde-3-phosphate Dehydrogenase,Cathepsin B endopeptidase and Actin-2.It is reported that Cathepsin B endopeptidase(CB)has important effect in hemoglobin degradation and can promote the growth of the schistosome.Therefore,the Cathepsin B endopeptidase(CB)may be an effective molecule of natural molecule vaccines against Schistosoma japonicum.So far no similar investigations about this molecule have been reported.Objective In order to ascertain whether CB can be used as target for the research of natural molecule vaccines against Schistosoma japonicum and whether it has stage specificity in vivo.Meanwhile,we use the fusion protein of the CB to screen the homologous ScFv antibody library to obtain the specific ScFv antibody.Methods 1.Total proteins were prepared from 32-days Schistosoma japonicum.After two-dimensional(2-D)gel electrophoresis, distinct protein spots from 2-D gels were isolated and analyzed by MALDI-TOF-MS.2.The target gene was obtained from the Sj cDNA library by means of PCR amplification with primers.The gene was then subcloned into the pcDNA3.0 vector;the recombinant plasmid was inoculated and tested their immunoprotective effect against schistosomiasis japonica in mice.3.The target gene was subcloned into the pQE30 vector and the recombinant plasmid was transformed into sensitized E.coli M15.The recombinant fusion protein was successfully expressed with IPTG and analyzed by SDS-PAGE and Western blot.Then we purify the fusion protein by His-tag affinity chromatograph.4.BALB/C mice were immunized with AWA and then extracted the total RNA from the spleens.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 pCANTAB5E and electroporated into competent E.coli TG1 cells.AWA single chain Fv phage display library was gained after the recombinant phagemids were rescued by reinfection of helper phage M13K07.Screen the ScFv antibody libraries against schistosomiasis japonica adult worm and UV-attenuated cercarie using the fusion protein mentioned above,in order to obtained the specific ScFv antibodies.Results 1.A total of 13 protein spots(which nearby the Sj31/32kDa)were detected in the 2-D gels.Three of them have highly homology with Sj glycerol-3-phosphate dehydrogenase,Sm cathepsin B endopeptidase and Sm actine-2.2.The recombinant plasmid of pcDNA3.0/SjCB was successfully constructed.PCR identifing,retriction enzyme digesting and nucleotide sequencing show the two recombinant plasmids have 99%homology. Compared with the control group,the worm,intestinal eggs,liver eggs and Intrauterine eggs reduction rates in the imago pcDNA3.0/SjCB vaccinated mice group were 38.99%,40.18%,43.24%,34.45%;and the cercarie pcDNA3.0/SjCB vaccinated mice group were 38.36%,39.30%, 44.87%,41.00%;there are no conspicuous differents between the two vaccinated groups.3.Also,the pQE30/SjCB recombinant plasmid was constructed and the SjCB recombinant fusion protein was expressed.PCR identifing, retriction enzyme digesting and nucleotide sequencing show the protein expressed and purified correctly.The recombinant protein could be recognized by anti-AWA mice serum and UV-attenuated cercarie serum but not by the normal mice serum.4.Schistosoma japonicum adult worm ScFv phage display library containing 4.3×10~8 clones,and the restructuring rate is 90%,also it has good diversity.Simply screened the specific ScFv antibodies against fusion protein.Conclusion 1.Successfully constructed the eukaryotic recombinant plasmid of imago pcDNA3.0/SjCB,cercarie pcDNA3.0/ SjCB and the prokaryotic recombinant plasmid of pQE30/SjCB,cercarie pQE30/SjCB.Nucleotide sequencing results shows that there is no evident different between the imago and cercarie cathepsin B endopeptidase.2.The animal experiment results show that pcDNA3.0/SjCB can induce partial immunoprotective efect against Schistosomiasis japonica in mice.The research identified and proved that SjCB is a potential vaccine candidate of natural molecule antigens against Schistosomiasis japonica.3.Construct the AWA scFv library successfully and obtain the specific scFvs against SjCB which may lay a foundation for the natural mulecule vaccine of schistosomiasis japonicum.