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In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 x 10(8). Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.
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Objective To compare a potential role of dendritic cells (DCs) and macrophages in inducing protective immunity against infection with Schistosoma japonicum. Methods DCs and macrophages were pulsed in vitro with soluble egg antigen (SEA) of S. japonicum. BALB/c mice were injected three times with DCs or macrophages, either antigen-pulsed or not,and challenged with 40 ± 2 cercariae of S. japonicum per mouse. Worms were collected 42 days later by portal perfusion of the mice and egg number of liver was calculated. To evaluate whether protective immunity had been induced by preparations of DCs or macrophages, the worm burden and fertility ( eggs per female per mouse liver) were compared between the groups of mice. The antibody level against SEA was detected by ELISA. Results With respect to mice injected with untreated cells, numbers of worms and eggs per female worms were significantly reduced in the groups of mice having received pulsed DCs (26. 3% and 37.9%, respectively), or pulsed macrophages (22. 0% and 30.7%). Untreated DCs and macrophages induced no significant effects. The antibody level against SEA rose in sera of all groups of mice up to 42 days after the challenge, but most pronounced in those immunized with pulsed DCs, although this was not significantly different from other groups. Conclusion The results suggest that the protective immunity against S. japonicum might be induced by DCs to a higher extent than by macrophages after in vitro pulsing with egg antigen.
ABSTRACT
In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.
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Objective To study the presence of iNOS in Schistosoma japonicum and demonstrate its distribution in different stages of this schistosome. Methods Cryostat sections for adult worms and paraffin sections for eggs in the liver of infected mouse, sporocysts and cercariae in snails were prepared, immunofluorescent test was performed to detect the presence of iNOS in adult worms, sporocysts, cercariae and miracidium, the distribution of this enzyme was observed in different stages of Schistosoma japonicum. Western blotting was used to further demonstrate the immunoreactivity to iNOS in adult worms. Results The results of immunofluorescent test showed that specific yellow- green fluorescence was mainly among subtugment of adult worms. Positive staining was also distributed on the surface of miracidium and its glands. For both sporocysts and cercariae, the majority of fluorescence was on their surface. Anti-iNOS antibody could recognize an apparent specific band in Western blotting of adult worm proteins, with a of Mr 210 000. Conclusion There is an expression of iNOS-like enzyme in Schistosoma japonicum.
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Objective To study the in vitro larvicidal activity of nitric oxide (NO) to the juvenile Schistosoma japoni-cum. Methods Macrophages were induced by LPS or LPS + IFN-? to produce NO, schistosomula obtained mechanically from cercariae were added to the medium with activated macrophages, the larvicidal activity was observed within 48 h . In order to further confirm the effect of NO, an inhibitor of iNOS,L-NNA (N?-nitro-L-arginine), was used to inhibit the production of NO, larvicidal activity was measured by the same methods and the difference of dead larvae ratio was compared between the inhibited and uninhibited groups. Results LPS and LPS + IFN-? can induce macrophages effectively, with the NO production of (109.96?3.70)?mol/L and (113.50?7.38) ?mol/L respectively, accordingly the larvicidal effect reached to 91.07% ?2.92% and 96.86%?2.36% respectively. This activity can be inhibited by L-NNA. NO production and dead larvae ratio were reduced significantly in the inhibited group than in the uninhibited one. Conclusion NO produced by activated macrophages can kill schistosomula of Schistosoma japonicum.
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Objective To investigate the effect of nitric oxide on the liver fibrosis of mice infected with Schistosoma japonicum during the early stage of schistosomiasis. Methods NMRI mice were treated with AMG-an iNOS inhibitor from day 23 post infection(p.i) to the sacrificed and the livers were collected at 38 days, 45 days p.i respectively. The expression and distribution of collagen typeⅠ, Ⅲ (ColⅠand ColⅢ) in liver tissues were investigated with the Picric acid-Sirius red staining techniques and differentiated with the polarization microscopy combining with picture analysis system. Hydroxyproline concentration in liver homogenate was measured by the biochemical methods. Results At 38 day p.i, the Picric acid-Sirius red staining showed that the hyperplasia of Col Ⅰ and ColⅢ in the livers of AMG treated mice increased significantly compared with the control livers, but there was no significant difference of hydroxyproline content between the two groups. At 45 days p.i, only the hyperplasia of Col Ⅰ in AMG treated group increased significantly compared with the control livers, and moreover, content of hydroxyproline of the inhibited mice was significantly higher than that of the control mice (P
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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
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Objective To study the association of human leukocyte antigen classⅡ(HLA-Ⅱ) alleles with genetic susceptibility and resistance to advanced hepatosplenic schistosomiasis japonica. Methods The allelic types of HLA-DRB1, DPA1, DQA1 and DQB1 were detected by polymerase chain reaction with sequence-specific primers (PCR-SSP) technique in 46 patients with advanced hepatosplenic schistosomiasis, characterized with extensive liver fibrosis. Another 43 subjects with chronic schistosomiasis were used as control. The statistical significance of differences in allelic frequencies was determined by ? 2 test. Results The frequencies of HLA-DRB1*04, DPA1*0103, DQA1*0601, DQB1*0201 in advanced patients were markedly higher than those in control group, while the frequencies of HLA-DQA1*0501 and DQB1*0601 in control group were higher than those in advanced patients. Conclusion The study indicated that HLA-DRB1*04, DPA1*0103, DQA1*0601 and DQB1*0201 showing a positive, statistically significant (P
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Objective To study the expression of inducible nitric oxide synthase (iNOS) in livers of mice infected with Schistosoma japonicum. Methods The livers of NMRI mice infected with S. japonicum were collected on day 21, 28, 38, 45 post infection(p.i.), total RNA of livers were extracted and kinetics of the mRNA expression of iNOS were detected by RT-PCR, the protein expression of iNOS was then confirmed by Western blotting and the distribution of iNOS in the infected liver was determined by immunohistochemical methods. Results The mRNA expression of iNOS was not detectable in the uninfected liver, iNOS mRNA expression was detected on day 21 p.i, the expression increased on day 28 p.i and peaked on day 38 p.i, then decreased slightly on day 45 p.i. Western blotting showed an iNOS expression in the livers only on day 38, 45 p.i. IFA test showed that the expression of iNOS was maily distributed in the granuloma of the livers. Conclusion S. japonicum infection can induce the expression of iNOS in a time-dependent manner in the liver of the host,and eggs may be the main factor in inducing the expression.