Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Bacterial Proteins ; Diphtheria Toxin/genetics* ; Escherichia coli/genetics* ; Humans ; Molecular Chaperones/genetics* ; Recombinant Proteins/genetics*

Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.
Angiogenesis Inhibitors ; biosynthesis ; Diphtheria Toxin ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Immunotoxins ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

OBJECTIVETo test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells.

METHODSThe diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed.

RESULTSThe beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells. Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ.

CONCLUSIONSelective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.

Diphtheria Toxin ; genetics ; Enhancer Elements, Genetic ; Genes, Immunoglobulin ; Genetic Therapy ; methods ; Humans ; Immunoglobulin kappa-Chains ; genetics ; Neoplasms ; therapy ; Peptide Fragments ; genetics ; Promoter Regions, Genetic ; Tumor Cells, Cultured

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.
Animals ; Antibodies, Bacterial ; genetics ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Chromatography, Affinity ; Diphtheria Toxin ; immunology ; Escherichia coli ; genetics ; Female ; Immunotoxins ; isolation & purification ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.

METHODSThe mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.

RESULTSThe expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.

CONCLUSIONSThe recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; therapy ; Diphtheria Toxin ; biosynthesis ; genetics ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Genomic Imprinting ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS); it serves as a model for the human multiple sclerosis (MS). In mice, EAE is mediated by T cells specific for various myelin basic proteins which migrate from the periphery to the CNS. In search of a way to prevent the induction and progression of EAE, we observed the effects of recombinant immunotoxin IP10-DT390 on blocking or eliminating the active T cells in the EAE model. In this paper is presented an experimental gene therapy-based model in which the mice were made resistant to EAE induction by plasmid DNA encoding recombinant immunotoxin that was injected into the leg muscles of mice. The new immuno-biological construct could selectively impair autoreactive T-cell homing while the duration of clinical signs is shorter, and the new construct would not affect other components of the immune response. These data demonstrated the effectiveness of the constructs in the treatment of EAE and suggested its usefulness in the treatment of other autoimmune diseases.
Animals ; Chemokine CXCL10 ; biosynthesis ; genetics ; therapeutic use ; Diphtheria Toxin ; biosynthesis ; genetics ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; pathology ; therapy ; Female ; Genetic Therapy ; Immunoglobulin Fragments ; biosynthesis ; genetics ; therapeutic use ; Immunotoxins ; genetics ; metabolism ; therapeutic use ; Mice ; Mice, Inbred C57BL ; Receptors, CXCR3 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; therapeutic use ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; T-Lymphocytes ; immunology ; Transfection

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.
Adjuvants, Immunologic ; Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Ehrlich Tumor ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Diphtheria Toxin ; genetics ; immunology ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Immunoglobulin G ; immunology ; Immunotherapy ; Mice ; Neovascularization, Pathologic ; Peptide Fragments ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tandem Repeat Sequences

OBJECTIVETo evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).

METHODSEAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.

RESULTSEAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.

CONCLUSIONThe immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.

Animals ; Antigens, CD19 ; analysis ; B-Lymphocytes ; cytology ; metabolism ; CD3 Complex ; analysis ; Chemokine CCL3 ; genetics ; metabolism ; Diphtheria Toxin ; genetics ; metabolism ; Disease Models, Animal ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; Female ; Flow Cytometry ; Immunoglobulin Fragments ; genetics ; metabolism ; Immunohistochemistry ; Immunologic Factors ; therapeutic use ; Immunotoxins ; therapeutic use ; Meninges ; chemistry ; pathology ; Mice ; Mice, Inbred C57BL ; Multiple Sclerosis ; drug therapy ; NIH 3T3 Cells ; Receptors, CCR5 ; analysis ; Recombinant Fusion Proteins ; genetics ; metabolism ; therapeutic use ; T-Lymphocytes ; cytology ; metabolism