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Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Sehistosorna japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The V_H and V_L genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F' induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Westem-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.
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Objective:To prepare human angiopoietin like protein 2(ANGPTL2) monoclonal antibody.Methods:The purified recombinant human ANGPTL2 was used to immunize BALB/c mice.Then,the mouse spleen cells were isolated and fused with mouse myeloma cells.After selecting with HAT medium and analyzing with ELISA assay,the hybridoma cell clones stably secreting human ANGPTL2 antibody were screened out.The monoclonal antibody against humain ANGPTL2 was purified by ammonium sulfate precipitation method from the supernatant liquid of hybridoma cell culture.Western blotting,Cell immunostaining,and immunohistochemisty staining were used to characterize the antibody.Results:A strain of hybridoma cell clones stably secreting human ANGPTL2 antibody was screened out.The ANGPTL2 monoclonal antibody prepared was proven useful. Conclusion:A monoclonal antibody against human ANGPTL2 was successfully prepared,which provide a basis for basic study of ANGPLTL2.
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<p><b>OBJECTIVE</b>To investigate the protective immunity induced by the anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum in mice.</p><p><b>METHODS</b>An orthogonal table L(16) (4 x 2(12)) was selected as the experimental design. Eight-week-old Kunming outbred mice (male and female) were randomly divided into 16 experimental groups and 2 control groups. Control groups were injected with SP2/0 ascites intraperitoneally. Mice from each group were infected with 100 +/- 2 cercariae of Schistosoma japonicum in the abdominal skin and were sacrificed on the thirtieth day postchallenge. Adult worms were recovered and counted by perfusion of the left ventricle-portal vein. The SP2/0 ascites injected mice were used as controls and the percentage of protection was calculated.</p><p><b>RESULTS</b>Active immunization of mice with NP30 could produce protection levels ranging from 22.36% to 50.46% depending on the different immunity protocols. The best immunization protocol was established from the results.</p><p><b>CONCLUSIONS</b>Active immunization with NP30 can induce a degree of protection to infection with Schistosoma japonicum cercariae and NP30 is a potential vaccine candidate against Schistosoma japonicum.</p>
Subject(s)
Animals , Female , Male , Mice , Analysis of Variance , Animals, Outbred Strains , Antibodies, Anti-Idiotypic , Allergy and Immunology , Therapeutic Uses , Antibodies, Monoclonal , Allergy and Immunology , Therapeutic Uses , Schistosoma mansoni , Allergy and Immunology , Schistosomiasis mansoni , Allergy and Immunology , Parasitology , Treatment Outcome , VaccinationABSTRACT
Objective] To amplify and sequence the light chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. [Methods] By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes enco ding FR1 and FR4 regions of immunoglobulin light chain variable regions, we designed a set of primers for amplification of V L gene. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNAs were extracted and used as templates for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger′s method. The V L gene was compared with GenBank and published mouse V L genes. [Results] The full length of V L gene was 318 bp. The V L gene was a member of mouse Ig ? light chain subgroup IV and generated from rearrangement of germ line V and J? 4 genes. The V L gene sequence has been registered by GenBank(accession No. AF206720). [Conclusion] The obtained V L gene was a potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum .
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Objective To study the molecular mechanism of apoptosis of cells in egg granuloma induced by anti-idiotypic antibody NP30 of Schistosoma japonicum.Methods BALB/c mice were randomly divided into two groups. The mice of the experimental group were immunized by injecting NP30 intraperitoneally for three times, while the mice of control group were injected normal saline intraperitoneally. The mice were sacrificed respectively on the 39th, 49th, 64th, 108th, 112nd day after challenge with schistosome cercariae. The expressions of apoptosis-related gene Bax, Bcl-2, death receptor Fas, FasL (Fas ligand) and c-Fos were examined by the S-P method of immunohistochemistry,and Bax, mRNA and Fas mRNA investigated by the in-situ hybridization. Results The expressions of Bax, Fas, FasL and c-Fos were positive in granuloma cells of both groups. The expressions of Bax and FasL in experimental group were higher than those in control group (P
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In this study the ability of the monoclonal anti-idiotypic antibody NP30 was tested as a substitute of diagnostic antigen in detecting antibody of Schistosoma japonicum from human sera by use of ELISA. The results showed that the seropositive rate was 98% with NP30 in the group of acute infection, which was comparable to 94% with gut associated antigens (GAA)and 98% with the soluble egg antigens (SEA); 87% with NP30 in the group of chronic infection which was comparable to 86% with GAA but lower than that of 98% with SEA. The false positive rate was about 3% for all three diagnostic antigens. The results also showed that the geometric mean titer (GMT) of antibody to NP30 was higher than that to GAA but lower than that to SEA in the acute infection group and the GMT of antibody to NP30 was lower than both those to GAA and to SEA in the chronic infection group,suggesting that the antibody to NP30 appeared earlier and decayed more quickly during the process of infection. The authors suggested that NP30 could be used for the diagnosis of schistosomiasis japonica.
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Objective To study the effects of the monoclonal anti idiotypic antibody NP30 active immunization on egg granuloma formation and hepatic fibrosis in Schistosoma japonicum infection. Methods ICR mice were actively immunized with NP30 100 ?g ?3 ip. every 10 days while the mice in control group were injected with SP2/0 ascites ip. simultaneously. After cercariae challenging,the mice were killed at the 4th, 8th,12th, 16th, 20th and 24th week, respectively.Mouse livers were removed and stained histochemically with VG and subjected to immunohistochemical assay of collagen type Ⅰ,Ⅲ and fibronectin(FN).The volume of egg granulomas and the content of collagen type Ⅰ,Ⅲ and FN were determined quantitatively by NYD 1000 Image Analysis System. Results The volume of egg granulomas in NP30 immunized group was much smaller than that of control group from the 12th week after cercariae challenge. The cellular components of egg granulomas in NP30 immunized group were significantly different from those of the control group,exhibiting two types of atypical egg granulomas were found.VG stain revealed that the average optical density of collagen in hepatic granulomas of experimental group was lower than that of control group.Immunohistochemical assay revealed that the contents of collagen type Ⅰ,Ⅲ and fibronectin in egg granulomas of experimental group were lower than those of control group. Conclusion NP30 vaccination may induce both cellular and humoral protective immunity to modulate egg granulomas and suppress liver fibrosis of schistosomiasis japonica.