AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFvantibody against esophageal cancer.METHODS: Total ...AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFvantibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed, esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coliHB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×10^6 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells,25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Ecal09 cells, but not to HeLa and NHEEC cells.Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup IV of immunoglobulin, and variable light (VL) gene from the κ chain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.展开更多
Objective To determine the distribution of HLA-B alleles in the Chinese Yi ethnic group and its association with HIV infection. Methods One hundred and six unrelated healthy HIV negative and 73 HIV positive Chinese Yi...Objective To determine the distribution of HLA-B alleles in the Chinese Yi ethnic group and its association with HIV infection. Methods One hundred and six unrelated healthy HIV negative and 73 HIV positive Chinese Yi ethnic individuals were typed by PCR-SSP. Results The frequency of alleles B*07, B*35, and B*46 were increased in HTV-1 -positive subjects, whereas the alleles B*55, B*44 and B*78 were absent in the HIV-infected persons studied. The B*46 allele was present in a significantly higher gene frequency among HIV-1-positive individuals (P=0.02, OR=3.32, 95% CI=1.13-9.78) compared with control subjects. Conclusion HLA-B*46 may be associated with its susceptibility to HIV-1 infections.展开更多
AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for es...AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P<0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P<0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.展开更多
Experimental autoimmune encephalomyelitis(EAE) is a good model for human multiple sclerosis(MS) research.However,there are some defects in the traditional models.Here,we improved the model by using the human myelin ba...Experimental autoimmune encephalomyelitis(EAE) is a good model for human multiple sclerosis(MS) research.However,there are some defects in the traditional models.Here,we improved the model by using the human myelin basic protein(MBP) as antigen.EAE was induced by immunization of female Wistar rats with human MBP.Compared with the traditional models,the new model was evaluated by clinical signs to pathological changes.The immune state of the model was assessed by the lymphocyte infiltrative response and levels of TNF-α,IFN-γ,IL-10.It was found that most of rats exhibited tail tone loss and hind-limb paralysis, also there were demyelination,infiltrative lymphocyte foci,“neuronophagia”in the cortex of cerebra and the white matter of spinal cords.PBMCs and spleen lymphocytes were strongly responsive to the stimulation of MBP and PHA.The levels of TNF-α and IFN-γ were altered with the severity of EAE.In the remitting phase, IL-10 was increased significantly.This study demonstrates that the animal model of EAE induced by human MBP bears resemblance to the features of human multiple sclerosis and promises to be a better model than ever before for the study of MS.Cellular & Molecular Immunology.2004;1(5):387-391.展开更多
基金Supported by the National Natural Science Foundation of China,No.30070291 andNo.30213914 Medical Scientific Foundation of Guangdong Province,No.A2000434
文摘AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFvantibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed, esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coliHB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×10^6 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells,25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Ecal09 cells, but not to HeLa and NHEEC cells.Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup IV of immunoglobulin, and variable light (VL) gene from the κ chain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.
基金This work was supported by the Special Funds for Major State Basic Research Program (973) of China (No.G1999054107) the National Key Technologies R & D Program of China during the 15th Five-years Plan Period (No. 2001BA705B02)
文摘Objective To determine the distribution of HLA-B alleles in the Chinese Yi ethnic group and its association with HIV infection. Methods One hundred and six unrelated healthy HIV negative and 73 HIV positive Chinese Yi ethnic individuals were typed by PCR-SSP. Results The frequency of alleles B*07, B*35, and B*46 were increased in HTV-1 -positive subjects, whereas the alleles B*55, B*44 and B*78 were absent in the HIV-infected persons studied. The B*46 allele was present in a significantly higher gene frequency among HIV-1-positive individuals (P=0.02, OR=3.32, 95% CI=1.13-9.78) compared with control subjects. Conclusion HLA-B*46 may be associated with its susceptibility to HIV-1 infections.
基金Supported by the National Natural Science Foundation of China,No.30210103904 and the Science and Technology Program of Guangdong Province,No.2003C30304
文摘AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P<0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P<0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.
文摘Experimental autoimmune encephalomyelitis(EAE) is a good model for human multiple sclerosis(MS) research.However,there are some defects in the traditional models.Here,we improved the model by using the human myelin basic protein(MBP) as antigen.EAE was induced by immunization of female Wistar rats with human MBP.Compared with the traditional models,the new model was evaluated by clinical signs to pathological changes.The immune state of the model was assessed by the lymphocyte infiltrative response and levels of TNF-α,IFN-γ,IL-10.It was found that most of rats exhibited tail tone loss and hind-limb paralysis, also there were demyelination,infiltrative lymphocyte foci,“neuronophagia”in the cortex of cerebra and the white matter of spinal cords.PBMCs and spleen lymphocytes were strongly responsive to the stimulation of MBP and PHA.The levels of TNF-α and IFN-γ were altered with the severity of EAE.In the remitting phase, IL-10 was increased significantly.This study demonstrates that the animal model of EAE induced by human MBP bears resemblance to the features of human multiple sclerosis and promises to be a better model than ever before for the study of MS.Cellular & Molecular Immunology.2004;1(5):387-391.
