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Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.
Subject(s)
Insecticides/metabolism , Bacillus thuringiensis , Endotoxins/pharmacology , Bacillus thuringiensis Toxins/metabolism , Hemolysin Proteins/pharmacology , Bacterial Proteins/chemistry , Plants, Genetically Modified/genetics , Pest Control, BiologicalABSTRACT
Both EAC-tumor associated gangliosides and its anti-idiotype antibody inhibited growth of this tumor significantly. Immuno-histological studies with von Willebrand Factor (vWF) antibody indicated that tumor angiogenesis as determined by expression of vWF decreased in tumors of mice, post-immunized with EAC-cell gangliosides as well as its anti-idiotype antibody. Infiltration of various immune cells of the host in the tumor correlated to some extent with tumor-growth inhibition. Apoptosis study using AnnexinV-FITC and propidium iodide indicated that tumor growth inhibition in mice post-immunized with EAC-gangliosides and its anti-idiotype antibody were due to enhanced apoptosis and cell death. Cell cycle analysis by FACS indicated that EAC-cell associated gangliosides and its anti-idiotype antibody were acting both at the M2 i.e. S and M3 i.e. G2/M phases of the cell cycle to arrest tumor growth.
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Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.
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Objective To compare the in vitro antitumor immune responses induced by bivalent bispecific anti-idiotype antibody G22-I50 and monovalent anti-idiotype antibody G22 and I50, and explore its possible mechanism. Methods Proteins G22-I50, G22, and I50 were induced and identified by Western blot and ELISA. Peripheral blood monoclear cells (PBMC) were isolated and stimulated with G22-I50, G22, and I50 anti-idiotype antibodies, respectively. MTT assay and LDH release test were employed to examine the proliferation and cytotoxicity of the PBMC. The levels of IFN-γ, IL-2, and IL-4 in the supernatant were detected by ELISA and changes of T lymphocyte subsets were determined by flow cytometry. Results Western blot showed that G22-I50, G22, and I50 had specific binding capabilities to FC2 (Ab1). The activities of G22-I50, G22, and I50 had recovered and these proteins could be used in the in vitro study. The proliferation and cytotoxicity of the PBMC stimulated with G22-I50 were significantly higher than those stimulated with G22 or I50, The level of IFN-γ and IL-2 in the culture supernatant of the PBMC stimulated with G22-I50 was higher than that in the G22 or I50 group, but the level of IL-4 did not increase.Compared with the G22 or I50 group, the proportion of CD4+ and CD8+ T cells and CD4+/CD8+ ratio significantly increased, and the proportion of CD4+CD25+ T cells significantly decreased in the PBMC stimulated with G22-I50. Conclusion G22-I50 has more potent immunogenicity and would enhance specific antitumor effect which might relate to improving PBMC proliferation, inducing the secretion of Th1 type cytokines, activating CD8+T cells, and suppressing the expression of CD4+CD25+ T cells.
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Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.
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Summary: The purpose of this study was to construct expression vectors of idiotype (Id) Smlg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id,and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by 1PTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ, in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. Coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.
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The unique antigenic determinants (Idiotype [Id]) of the immunoglobulin expressed on a given B-cell malignancy can serve as a tumor-specific antigen for active immunotherapy. Therapeutic vaccines targeting the tumor-specific idiotype have demonstrated promising results against lymphomas in phase I/II studies and are currently being evaluated in phase III randomized trials. Additional vaccine therapies being developed include those based on DNA, dendritic cells, gene-modified tumor cells. It is hoped that immunotherapeutic agents, used in tandem or in combination, may in the future allow effective treatment of lymphoid malignancies and delay or even replace the need for conventional cytotoxic therapies.
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Objective To construct,express and identify the anti-idiotypic antibody single chain variable fragment(scFv) against Edwardsiella tarda.Methods By using RT-PCR method,the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody(mAb) 1E11 against Edwardsiella tarda were cloned and joined with a(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH was constructed.It was then cloned into vector plasmid pET-28a,expressed in Escherichia coli BL21(DE3),and confirmed by SDS-PAGE,Western blot and ELISA.Results The recombinant scFv could be expressed in E.coli BL21(DE3) in a fusion protein pattern.The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded.The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv had the binding activity to the antigen.Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21(DE3),providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.
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Objective:To analyze whether the DNA vaccine against the CDR3 regio n of immunoglobulin heavy chain of the human B-cell lymphoma could elicit the s p ecific idiotypic antibody,the authors established a model with the human B-cell lymphoma c ell line Namalwa.The CDR3 gene fragment of Namalwa membranous immunoglobulin hea vy chain was amplified.The sequenced CDR3 fragment was used as the antigen gene to construct the DNA vaccine plasmid. Methods:The authors acquired the CDR3 gene fragment using Ig superfamily primers by means of RT-PCR and the murine monocyte chemot ic protein(MCP-3) as the adjuvant molecular.The fused gene fragment of CDR3 and MCP-3 was obtained by recombinant PCR and then cloned into the eukaryonic plas mid vector pcDNA3.1 to construct the DNA vaccine plamid.Then the vaccine plasmid was transiently expressed in the eukaryonic cell COS-7. Results:The authors acquired th e DNA vaccine plasmid in which the mIgCDR3 of the Namalwa cell was used as the a ntigen gene by the molecular biology. Conclusion:The transient transfection assa y proved that the recombinant plasmid could express in eukaryonic cells in right way.They have constructed an expression plasmid containing fused MCP3-CDR3 seq uen ce which could be used in further study of DNA vaccine against B-cell lymphoma in vivo. [
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Anti-idiotype antibody (anti-id Ab) which recognizes idiotope in the variable region of immunoglobulin (Ig) can regulate Ab production by B cells in vivo and in vitro. Although it has been reported that anti-id Ab can suppress IgM production by lymphocytes or hybridoma cells without suppression of cell proliferation, the regulatory mechanism of anti-id Ab is not completely understood. We studied the effects of anti-id Ab on the production of IgG class anti-DNA Ab by hybridoma cells, on the proliferation of cells, and on the transcription levels of Ig genes. In contrast to suppressive effect of anti-id Ab on the production of IgM previously reported by others, stimulatory effects of anti-id Ab on the production of IgG by hybridoma cells as well as the proliferation of these .cells were observed. However, little effect of anti-id Ab on the transcription levels of Ig genes was observed. These results suggest that anti-id Ab can increase Ab production by stimulation of cell proliferation. Furthermore, these results suggest that the effect of anti-id Ab on the production of Ab may be determined by the difference in class of Ab produced by hybridoma cells following the treatment with anti-id Ab.
Subject(s)
Antibody Formation , B-Lymphocytes , Cell Proliferation , DNA , Genes, Immunoglobulin , Hybridomas , Immunoglobulin G , Immunoglobulin M , Immunoglobulins , LymphocytesABSTRACT
Two hybridomas (TD_1, TD_2)secreting monoclonal anti-idiotypic antibodies (anti-ld) againstanti-HBs were established by fusing the myeloma cells, IR983F, with the spleen cells of LOU/Crat which had been immunized with mouse monoclonal anti-HBs to the a determinates and syn-genic rat polyclonal anti-HBs. TD_2(Ab_2)showed the ability to inhibit the binding of horse anti-HBs with HBsAg and was also neutralized by anti-HBs from various origins. Ab_2 can induce an-ti-anti-ld (Ab_3)when injected it into LOU/C rats introperitoneally. Ab_3 can react specifically withHBsAg. These data indicate that TD_2 monoclonal anti-ld against anti-HBs are the anti-ld bearingthe internal image of HBsAg epitope that could mimic HBsAg and possess the immunogenicity ofHBsAg.
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Rabbit anti-TSH anti-idiotypic antiscra were raised by immunization with auti-TSH antibody. The antiserum from rabbit No. 551 was found to have the activity of inhibiting the binding of TSH receptor on thyroid cell membrane and 125I-TSH, that is, TBII (TSH-binding inhibitor immunoglobulin) positive. This antiserum was also shown to inhibit cAMP release stimulated by 100 mU/L bTSH. The characteristics of No. 551 antiserum were similar to of patients with myxedema.
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This article reported a method of anti idiotype antibodies to idiotype in patientswith multiple myeloma.SmIg and CIg of peripheral blood lymphocytes of patientswith multiple myeloma were examined by using anti idiotype antibodies. The result revealed that(1)the anti idiotype antibody only reacted to M pro-tein and serum of the patient,but not to M proteinemia and normal Ig.(2)SmIg andCIg of some B cell reacted to the antiidiotype antibodies,it has been suggestedthatthe abnormal colon of multiple myeloma formed in B cell
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The anti-idiotypic antibodies Specific for anti-HBs were prepared by Immunizing New Zealand white rabbits with anti-HBs according to the method of Kennedy. The serum was first purified through IgG of normal human-Sepharose 4B affinity Column.The anti-idiotypic Specificity of the antibodies obtained was identified with ELISA or RIA inhibition test.The resultshowed that the anti-idiotypic antibodies were directed at the idiotypic determinants on anti-HBs,and no cross reaction between anti-idiotypic antibodies and normal human IgG.