OBJECTIVETo construct a lentiviral expression vector of human carcinoembryonic antigen (CEA), and identify its expression in dendritic cells (DCs).

METHODSHuman CEA-encoding sequence was amplified, purified, ligated with lentiviral vector plasmid pLentiGFP and verified by sequencing. The verified recombinant vector plasmid (pLentiGFP-CEA), the packaging plasmid p 8.2 and pVSV-G were transfected into 293T cells by Lipofectamine(TM) 2000 reagent. The supernatant of the cultured 293T cells was collected to infect the DCs. The expression of CEA in the transfected DCs was assayed by RT-PCR and Western blotting.

RESULTSCEA lentiviral vector was highly expressed in the transfected DCs as observed using fluorescence microscope 48 h after the the transfection. The human CEA gene was successfully amplified by RT-PCR with a length of about 2100 bp. Western blotting also showed CEA expression in the transfected DCs.

CONCLUSIONThe human CEA lentiviral expression vector has been successfully constructed and the functional CEA protein can be expression in the transfected DCs. This facilitates further studies of the function of CEA at the molecular level.

Carcinoembryonic Antigen ; biosynthesis ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection

OBJECTIVETo observe the changes in the activity of dendritic cells (DCs) after carcino-embryonic antigen (CEA) gene transfection mediated by recombinant adeno-associated virus type2 (rAAV) and tumor cell lysate.

METHODSImmature DCs isolated from peripheral blood monocytes of HLA-A11-positive healthy volunteers were infected with the rAAV carrying CEA gene or loaded with tumor cell lysate. The surface markers of the DCs such as CD40, CD 1alpha, and CD86 were analyzed by flow cytometry. Interleukin-12 (IL-12) in the supernatants of DCs and interferon-gamma (IFN-gamma) released by the cytotoxic T lymphocytes (CTLs) were determined by ELISA detection kit. The specific killing activity of CTL against LoVo cells was assessed by MTT assay.

RESULTSThe DCs following antigen loading with the two methods both highly expressed CD40, CD86 and IL-12, and induced specific CTL that specifically recognized and killed LoVo cells, but the killing effect resulting from rAAV infection of the DCs was much better than that induced by tumor cell lysate loading.

CONCLUSIONBoth methods of antigen loading can induce mature DCs from peripheral blood monocyte cells, but rAAV infection of the DCs can be more effective than tumor cells lysate loading. DCs infected with rAAV may have the potential to serve as an adjuvant immunotherapy for patients with colorectal carcinoma.

B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; biosynthesis ; immunology ; Carcinoembryonic Antigen ; genetics ; Cell Line, Tumor ; Colorectal Neoplasms ; therapy ; Dendritic Cells ; immunology ; metabolism ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Interleukin-12 ; metabolism ; Transfection

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology

Eight saporin peaks were obtained from the purification of seed extracts of Saponaria officinalis L. Saporin peak No. 6 (SAP-6) showed the highest activity in the inhibition of protein synthesis (98%) in an in vitro translation study. An immunotoxin (IT) was prepared from SAP-6 conjugated to a monoclonal anti-CEA antibody 26/5/1 (mab B) using N-succinimidyl pyridyl dithiopropionate (SPDP) and 2-iminothiolane as a cross linker. Under thermal stability study by a DSC (differential scanning calorimetry), the IT showed a denature temperature of 75 degrees C. In in vitro translation studies, the purified IT showed the same activity as SAP-6 at 10(-7) M and 10(-9) M protein concentration at 0, 30 and 60-min incubation effects with mab B and SAP-6 not conjugated at 24-hr incubation periods on human promyelocytic cell line HL 60 and on human colon adenocarcinoma cell lines which were SW 403, LoVo and LS 174 T. SAP-6, mab B and IT had no cytotoxic effect on HL-60. The IT showed a higher cytotoxic effect than SAP-6 in CEA-positive cell lines. The IT demonstrated the highest cytotoxic effect of 51% inhibition of control at 10(-7) M on the LS 174 T.
Antibodies, Monoclonal/*therapeutic use ; Antineoplastic Agents, Phytogenic/administration & dosage/*pharmacology ; Carcinoembryonic Antigen/biosynthesis/*immunology ; Colonic Neoplasms/*pathology/therapy ; Hot Temperature ; Humans ; Immunotoxins/*therapeutic use ; *N-Glycosyl Hydrolases ; Plant Proteins/administration & dosage/*pharmacology ; Ribosome Inactivating Proteins, Type 1 ; Tumor Cells, Cultured/drug effects