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Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.
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To observe the dynamic changes of splenocyte proliferation ,subsets and apoptosis in mice immunized with re-combinantBifidobacteriumbifidum(pGEX-Sj26GST)ofSchistosomajaponicum,themiceweresubcutaneously(SCgroup) and intranasally (IN group) immunized ,respectively .Four mice from each group were sacrificed in every 2 wk during 0-20 wk after immunization .Splenocyte proliferation was investigated by MTT colorimetric assay ,subsets of CD+4 and CD+8 T cells and apoptosis of splenocytes by FACsort flow cytometry .In SC group ,unstimulated and stimulated with S jAWA ,the level of splenocyte proliferation significantly increased at 4-20 wk after vaccination and increased markedly at 4-18 wk stimulated with ConA ,both of which peaked at 8 wk;in IN group ,the proliferation level of splenocyte cultured with SjAWA and ConA signif-icantly increased during the 4-18 wk ,2-10 wk and 14-18 wk ,2-8 wk and 12-18 wk ,respectively ,and all reached the maximum at the 4 wk after immunization (P＜0 .01 or P＜0 .05) .CD+4 subsets increased obviously during 2-14 wk ,2 wk and 6-16 wk re-spectively ,and reached the peak at 8 wk (P＜0 .01 or P＜0 .05) in both group ,while CD8+ subsets rose lightly during 2-20 wk in both group ,and reached the maximum at 8 wk (SC group) and 6 wk respectively (P>0 .05) .Whether unstimulated or stimulated with ConA ,the level of splenocyte apoptosis of which remarkably increased at 2-4 wk and 2-6 wk separately in SC group ,and both peaked at 2 wk (P＜0 .01 or P＜0 .05 );in IN group ,the level of splenocyte apoptosis all increased at 4 wk and reached the maximum at the same time .In summary ,by inducing the proliferation of splenocytes ,increasing CD4 + T cells and decreasing splenocyte apoptosis ,the rBb (pGEX-Sj26GST ) vaccine plays a critical role in the protective immune response .
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Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity.
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Objective To construct and express the recombinant plasmid pET32α-Sj26GST of Schistosoma japonicum(sj)in Escherichia coli(E.coli)B121(DE3).Methods The total RNA was extracted from sj adult worms by ultrasound-breaking,Sj26GST antigen gene was amplified by RT-PCR from the total RNA,then cloned into prokaryotic expression plasmid pET32α(+) and transformed into E.coli B12(DE3)to construct pET32α-Sj26GST;BL21(pET32α-Sj26GST)WaS induced with isopropyl-β-D-thiogalactopyranosid(IPTG),and the expressed products were analyzed and identified by SDS-PAGE and Western blot.Results The 676 bp Sj26GST gene was successfully amplified by RT-PCR and cloned into pET32α(+)by restriction analysis and PCR identification,the recombinant plasmid pET32α-Sj26GST was successfully constructed;the relative molecular mass of the expressed recombinant protein was approximately 49×103 by SDS-PAGE,and the amount of the expressed protein was 24%of the total bacterial proteins;the fusion protein could be recognized by sera from rabbits infected with sj by Western blot.Conclusions The recombinant plasmid pET32α-Sj26GST is successfully constructed and highly expressed in E.coli in fused form with Trx-tag and His-tag,and the expressed fusion protein shows specific antigenicity.
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To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj14-Sj26 that contains fatty binding protein (Sj14), GST (Sj26) and paramyocin (Sj97) that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was trans-fected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and pIRES-Sj97-Sj14-Sj26 plasmid DNA, pIRES-Sj14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA,plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh.Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the pIRES-Sj97-Sj14-Sj26 group as compared with the plRES blank vector, normal saline and pIRES-Sj26 groups (P<0.01) and the pIRES-Sj14-Sj26(P<0.05). Single splenocyte suspension was prepared to detected the level of IFN-γ by ELISA and the lymphocyte stimulating index (SI) by MTT SI was significantly higher of in the pIRES-Sj97-Sj14-Sj26 group than in other groups (P<0.01), while the IFN-γ, level was significantly higher the pIRES-Sj97-Sj14-Sj26 group than in pIRES blank vector and normal saline groups (P<0.01), but no significant differences were found when compared with pIRES-Sj14-Sj26 and pIRES-Sj26 groups. Flow cytometery showed that the percent-ages of CD4+ and CD8+ T cells were much higher in the pIRES-Sj97-Sj14-Sj26 group (P<0.01,P<0.05). It was concluded that pIRES-Sj97-Sj14-Sj26 vaccine may induce stronger immune response in BALB/c mice.
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In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sjl4 or pVAC-Sj26 only to get two gene fragments including Sjl4 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES,resulting in another new recombinant plasmid pIRES-Sj26-Sj 14. The expression of Sj14 and Sj26genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and pIRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
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Aim To study the effects on T lymphocyte subsets in spleen of mice immunized with recombinant BCGSj26GST. Methods Inthe first experiment, BALB/c mice were immrnized subcutaneously by 106 and 108 CFU BCG-Sj26GST respectively. ALLthe mice were artifically challenged with cerariae of Schistosoma japonicum on 8weeks after immunization, six after challenge, the mice were killed, and the spleens were removed, cells were prepared seperately and were analysed by immunofluorescence with conjugated monoclonal antibodies in a FACsort cytofiuorimeter at 523 nm, PBS treated mice were serwed as control. In the second experiment, after immunized subcutanenously or inntraveously by 106 CFU vaccine, 4 mice were ranndonly killed to separate spleens on 0wk, 4wk, 8wk, 10wk, 14wk and 16wk after immurization, the percentage of CD+4 and CDs+ subsets was analysed as above. Results In the first experiment, CD+4 subsets increased renarkably, but CD+8 subsetsdid slightly by immunization with the vaccine against challenge with S.j. Cercariae, in the second experiment, the dynamic observation showed that in the subcutaneous and intravenous group, CD+4 subsets increased obviously since 10wk and 8wk respectively, CD8+ subsets hadno obvious changeover 16 weeks of observation , in the subcutaneous groupbuttheCD8+ subsets rose lightly on 4-16wk in the intravenous group. Conclusion On the basis of this study it's suspected that CD+4 subsets might play an important role in the protective immunity induced by the vaccine
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Objective To construct a multivalent DNA vaccine.\ Methods\ The multivalent DNA vaccine candidates pBK Sj26 Sj23,pBK Sj32 Sj23 were constructed based on the plasmids pBluescript Sj26,pBluescript Sj32 and pBluescript Sj23 with three pairs of specific primers using DNA recombinant technique. In the primers, a synthetic linker sequence encoding a peptide was designed,and the antigen genes Sj26 and Sj23,Sj32 and Sj23 were then ligated. After identification, the quadriceps muscle of mice were immunized with the multivalent antigen genes. Four weeks after immunization, the multivalent antigen genes were present in the muscular tissue of mice by PCR.\ Results\ The eukaryotic plasmids including multivalent antigens of S.japonicum were constructed successfully, and the plasmids including multivalent antigen gene could be stably existing in the muscle tissue of mice and the multivalent antigens could be expressed in the muscle tissue cells of mice.\ Conclusion\ A multivalent S.japonicum DNA vaccine has been established.
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Objective To explore the mechanism of protective immunity against Schistosoma japonicum infection induced by Sj26 gene transfected dendritic cell(DC).Methods 48 BALB/c mice were divided randomly into 4 groups with 12 each.The mice were injected through auricle for three times with Sj26 gene transfected DC(Group A),pcDNA3 transfected DC(Group B),untreated DC(Group C) and RPMI-1640(Group D) respectively,and challenged with 40?2 cercariae of S.japonicum per mouse 2 weeks after the last immunization.Sera from mice were examined for IgG antibody,IFN-? and IL-4 by ELISA.Western blot was used for detecting specific anti-Sj26 IgG antibody.The production of IFN-? and IL-4 in the supernatant of spleen cells stimulated with soluble egg antigen(SEA) and ConA was quantified by sandwich ABC-ELISA.The proliferation of spleen cells were measured with MTT method.Results IgG antibody increased significantly in the mice of group A at 2 weeks after the last immunization(absorbency A491=0.117),higher than that of group B(A491=0.061) and group C(A491=0.058)(P