The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain canc...The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.展开更多
This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragment...This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.展开更多
Niobium-doped indium tin oxide （ITO：Nb） thin films are fabricated on glass substrates by radio frequency （RF） magnetron sputtering at different temperatures. Structural, electrical and optical properties of the f...Niobium-doped indium tin oxide （ITO：Nb） thin films are fabricated on glass substrates by radio frequency （RF） magnetron sputtering at different temperatures. Structural, electrical and optical properties of the films are investigated using X-ray diffraction （XRD）, atomic force microscopy （AFM）, ultraviolet-visible （UV-VIS） spectroscopy and electrical measurements. XRD patterns show that the preferential orientation ofpolycrystalline structure changes from （400） to （222） crystal plane, and the crystallite size increases with the increase of substrate temperature. AFM analyses reveal that the film is very smooth at low temperature. The root mean square （RMS） roughness and the average roughness are 2.16 nm and 1.64 nm, respectively. The obtained lowest resistivity of the films is 1.2 × 10^4 Ω-cm, and the resistivity decreases with the increase of substrate temperature. The highest Hall mobility and carrier concentration are 16.5 cmVV.s and 1.88× 10^21 cm^-3, respectively. Band gap energy of the films depends on substrate temperature, which is varied from 3.49 eV to 3.63 eV.展开更多
To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verif...To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.展开更多
基金This work was supported by funds of Natural Science of Scientific Committee and Educational Committee of AN-HUI Province respectively, and Hi-tech Research and Development Program ("863" Program).
文摘The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.
文摘This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.
文摘Niobium-doped indium tin oxide （ITO：Nb） thin films are fabricated on glass substrates by radio frequency （RF） magnetron sputtering at different temperatures. Structural, electrical and optical properties of the films are investigated using X-ray diffraction （XRD）, atomic force microscopy （AFM）, ultraviolet-visible （UV-VIS） spectroscopy and electrical measurements. XRD patterns show that the preferential orientation ofpolycrystalline structure changes from （400） to （222） crystal plane, and the crystallite size increases with the increase of substrate temperature. AFM analyses reveal that the film is very smooth at low temperature. The root mean square （RMS） roughness and the average roughness are 2.16 nm and 1.64 nm, respectively. The obtained lowest resistivity of the films is 1.2 × 10^4 Ω-cm, and the resistivity decreases with the increase of substrate temperature. The highest Hall mobility and carrier concentration are 16.5 cmVV.s and 1.88× 10^21 cm^-3, respectively. Band gap energy of the films depends on substrate temperature, which is varied from 3.49 eV to 3.63 eV.
基金the Scientific Research Project of Shanghai Municipal Health Bureau (2008Y034)the Natural Scientific Research Project of Shanghai(05ZR14086)
文摘To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.
