Construction, Screening Of Single-chain Fv Antibody Library Against Immature Egg Of Schistosoma Japonicum And Application In Vaccine Research

Schistosomiasis remains to be a serious zoonosis.It is well known that mature egg of Schistosoma is the major causative agent to the development of schistosomiasis and is responsible for the transmission and prevalence of this disease.So that searching for vaccines against Schistosoma japonicum is chiefly focused on anti-embryonation and anti-fecundity immunity.With years of effort,our laboratory has proved that a strong anti-embryonation and anti-fecundity immunity in host can be induced after being immunized with soluble immature egg antigen(SIEA)of Schistosoma japonicum.Protective humoral immunity induced by SIEA is the main mechanism of the protective effects.SIEA immune serum can react with the vitellaria and the lining membrane tissue of the gut lumen of female worms as well as with the embryo of immature eggs,which inhibited the embryo development and the fecundity of female worms markedly.We also found in further studies that 26-28kDa components extracted from SIEA are the major antigens inducing the protective humoral immune response.But the difficult in purifying and preparing the natural molecular vaccine,SIEA26-28kDa in quantity hampered the application of the natural molecular vaccine.So that,obtaining highly specific SIEA26-28kDa antibody may be a means of solving the problem. As a probe,the specific antibody can be used to screen,analyze and identify the coding genes of the natural molecular candidate vaccine, SIEA26-28kDa.Phage display technique,an increasingly important tool in biotechnology,provides an effective means for us in obtaining single-chain Fv antibody possessing the same antigen binding activity and specificity as complete antibody rapidly.Objective In this research,we aimed to obtain specific single-chain Fv antibody against SIEA26-28kDa,the natural molecular candidate vaccine and get the coding genes of SIEA26-28kDa molecules by screening the cercaria cDNA library of Schistosoma japonicum with the specific scFv.Methods1)Single-chain Fv antibody library against SIEA(soluble immature egg antigen)of Schistosoma japonicum was constructed by phage display technology.BALB/C mice were immunized with SIEA and total RNA was extracted from the spleens of mice.First strand cDNA was synthesized from the total RNA template.And variable heavy(VH)and variable light(VL)genes were amplified from first-strand cDNA using VH and VL degenerated primers respectively.Then VH and VL DNAs were assembled to yield the single-chain variable fragments randomly by SOE-PCR(splicing by overlap extension).Then the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent E.coli TGl cells.Phage display SIEA scFv library was obtained after the recombinant phagemids were rescued by helper phage M13K07.The repertoire,recombination rate and diversity of the phage display library were detected.2)Recombinant phages carrying scFv inserts were subjected to four rounds of enriching for reactivity against SIEA26-28kDa.Clones were detected by colony lift assay or enzyme-linked immunosorbent assay (ELISA)to obtain positive clones against SIEA26-28kDa.Positive clones were used to infect E.coli HB2151 to produce soluble scFv antibodies. SDS-PAGE and Western blot analysis were conducted to identify the expression level,molecular weight of soluble scFv and its binding activity and specificity with SIEA26-28kDa respectively.3)In order to elevate the expression of soluble SIEA26-28kDa scFv,the SIEA26-28kDa scFv coding gene was subcloned to prokaryotic expression vector PET32a to construct recombinant expression plasmid PET32a/scFv.On the other hand,in order to obtain scFv tagged with EGFP,enhanced green fluorescence protein(EGFP)coding gene was amplified and subcloned to PET32a/scFv to construct recombinant expression plasmid PET32a/EGFP-scFv.Transformed the two recombinant expression plasmids into E.coli BL21(DE3)and expressed in prokaryotic system.Expression levels,molecular weights, antigen-binding activities and specificities of Trx-scFv and Trx-EGFP-scFv fusion proteins were analyzed by SDS-PAGE and Western blot analysis.Tissue section of adult worms and eggs of Schistosoma japonicum were incubated with Trx-EGFP-scFv fusion protein,and the GFP singals were observed by fluorescent microscope to evaluate the targeting ability of the specific scFv.4)The cercaria cDNA library of Schistosoma japonicum was screened by using the highly-expressed specific SIEA26-28kDa scFv as a probe. Positive clones were sequenced and homology analysis of nucleic acid and deduced amino acid sequences were processed by softwares on line such as BLASTn and BLASTp in NCBI(The National Center for Biotechnology Information,http://www.ncbi.nlm.nih.gov).5)The coding sequences of these corresponding genes encoding SIEA26-28kDa were amplified from Schistosoma japonicum cercaria cDNA library and were subcloned to prokaryotic expression plasmid pQE30 to construct recombinant expression plasmids.Transformed the recombinant expression plasmids into E.coli M15 and expressed in prokaryotic system.SDS-PAGE and Western blot analysis were conducted to identify the expression levels,molecular weights and immunoreactivity of these fusion proteins.6)The coding sequences of these corresponding genes encoding SIEA26-28kDa were amplified from Schistosoma japonicum cercaria cDNA library and subcloned to eukaryotic expression plasmid pcDNA3 to construct recombinant expression plasmids.Kuming mice were grouped as follows:NS group,pcDNA3 plasmid group,Eukaryotic recombinant plasmid pcDNA3/SjRPS4 group,Eukaryotic recombinant plasmid pcDNA3/SjRPL7 group.These mice were immunized with naked eukaryotic plasmids or NS respectively,and then challenged with cercariae of Schistosoma japonicum.Immunoprotection against Schistosomiasis japonicum in mice was evaluated by worm reduction efficacy,liver eggs reduction efficacy,intestine eggs reduction efficacy and intrauterine egg reduction efficacy.Results1)A single-chain Fv antibody library against soluble immature egg antigen(SIEA)of Schistosoma japonicum was constructed.The repertoire of the phage display antibody was about 2.27×10~7.Expected scFv fragments about 780bp were all amplified from the randomly picked clones.The restriction enzyme digestion patterns of the randomly picked clones by BstN I was diverse.2)A specific scFv against SIEA26-28kDa was obtained by colony lift assay.The relative molecular weight of the specific scFv was about 32kDa.But the soluble expression level of SIEA26-28kDa scFv was relatively low.When SIEA was incubated with soluble SIEA26-28kDa scFv,only a single and distinct immunoreactive band at about 26-28kDa was detected.3)Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids PET32a/scFv and PET32a/ EGFP-scFv were successfully constructed.Soluble Trx-scFv and Trx-EGFP-scFv fusion proteins whose molecular weights were identical with the theoretical MWs,were proved to be efficiently expressed and still retained the binding activity and specificity with SIEA26-28kDa. Immunofluorescent localization of SIEA26-28kDa scFv tagged with EGFP showed that GFP signals were enriched to the embryonic cells of immature eggs,the genital system and lining membrane tissue of the gut lumen of female worms.But only weak fluorescent signal was observed in the lining membrane tissue of the gut lumen of male worms.4)Two corresponding genes encoding SIEA26-28kDa were obtained by screening the cercaria cDNA library of Schistosoma japonicum with the specific scFv.They are ribosomal protein S4 and ribosomal protein L7 of Schistosoma japonicum.5)Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pQE30/SjRPS4,pQE30/SjRPL7 were successfully constructed.The recombinant fusion proteins,whose molecular weights were identical with the theoretical MWs,were efficiently expressed and could be recognized by specific SIEA26-28kDa scFv and Schistosoma japonicum-infeeted mouse serum.But could not be recognized by normal mouse serum.6)Restriction enzyme digestion,PCR and sequencing results confirmed that the recombinant expression plasmids pcDNA3/SjRPS4, pcDNA3/SjRPL7 were successfully constructed.Compared with the control group,the worm and the egg reduction rates were all statistically significant in pcDNA3/SjRPS4,pcDNA3/SjRPL7 groups.Conclusion1)A high quality SIEA single-chain Fv antibody library was constructed successfully.The repertoire,recombination rate and diversity of the SIEA scFv library are good enough in facilitating the high-flux screening of specific scFvs against candidate vaccines.2)Specific scFv against SIEA26-28kDa,the natural molecular vaccine against schistosomiasis was obtained by colony lift assay quickly.3)Specific SIEA26-28kDa scFv not only possesses the same binding activity and specificity,targeting ability as the complete specific SIEA26-28kDa antibody,but also can be prepared in large scale by genetic engineering.So that specific SIEA26-28kDa scFv can replace the place of the complete SIEA26-28kDa-specific antibody in the following research.4)The specific SIEA26-28kDa scFv was used as a probe in screening cercarie cDNA library of Schistosoma japonicum to obtain two coding genes of the natural molecular candidate vaccine,SIEA26-28kDa.They are ribosomal protein S4 and ribosomal protein L7.5)The SjRPS4-6×His and SjRPL7-6×His prokaryotic recombinant fusion proteins possess satisfactory immunoreactivity.Eukaryotic recombinant plasmids pcDNA3/SjRPS4 and pcDNA3/SjRPL7 could induce significantly protective immunity against Schistosoma japonicum.Higher egg reduction percentages than worm reduction rates in all groups suggest that SjRPS4 and SjRPL7 could play a role mainly in anti-embryonation and anti-fecundity immunity against Schistosoma japonicum.They are potential candidate vaccines against schistosomiasis from SIEA26-28kDa.