Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were s...Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.展开更多
Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant p...Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.展开更多
基金supported by the National Natural Science Foundation of China (Grant No.30740013)the Key Laboratory for Genetics and Breeding in Forestry Trees and Ornamental Plants,Ministry of Education (03-05)
文摘Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.
基金Supported by the National Natural Science Foundation of China (30600665)the Natural Science Foundation Project of CQ CSTC (CSTC, 2008BB5107)+1 种基金the Youth Scientific Research Foundation of Third Military Medical University (06XG048)the Open Project Program of the State Key Laboratory of Trauma, Burns and Combined Injury (2006A-3)
文摘Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.
