Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revea...Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.展开更多
Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em&g...Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em> (<em>FUS3</em>) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of <em>FUS3</em> gene (<em>TaFUS3</em>) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated <em>TaFUS3-3A</em>, <em>TaFUS3-3B</em> and <em>TaFUS3-3D</em>, were first isolated from common wheat. The transcription of three <em>TaFUS3</em> genes in seed development and germination process was detected.<em> TaFUS3-3B</em> and<em> TaFUS3-3D</em> had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of <em>TaFUS3-3A </em>was not detected. Silencing of <em>TaFUS3</em> in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the <em>TaFUS3</em>-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of <em>TaFUS3</em> in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that<em> TaFUS3</em> plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.展开更多
To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression...To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.展开更多
The apoE cDNA was amplified from four months old fetus liver tissue mRNA by RT PCR. It was inserted into the TOPO ○R vector, whose sequence is the same as that reported. The right apoE cDNA was inserted into pQE30 ve...The apoE cDNA was amplified from four months old fetus liver tissue mRNA by RT PCR. It was inserted into the TOPO ○R vector, whose sequence is the same as that reported. The right apoE cDNA was inserted into pQE30 vector to construct pQE30 apoE recombinant expressed plasmid, which was expressed as a histidine tag fusion protein in E.coli strain BL21(DE3) with higher level. The purified protein was gained by Ni NTA column. SDS PAGE showed that 34 kD protein was expressed. Western blot analysis showed a positive band at 34 kD.展开更多
基金ThisprojectwassupportedbyagrantfromtheHubeiNaturalScienceFoundation (No .2 0 0 2ABA0 0 4 )
文摘Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.
文摘Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em> (<em>FUS3</em>) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of <em>FUS3</em> gene (<em>TaFUS3</em>) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated <em>TaFUS3-3A</em>, <em>TaFUS3-3B</em> and <em>TaFUS3-3D</em>, were first isolated from common wheat. The transcription of three <em>TaFUS3</em> genes in seed development and germination process was detected.<em> TaFUS3-3B</em> and<em> TaFUS3-3D</em> had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of <em>TaFUS3-3A </em>was not detected. Silencing of <em>TaFUS3</em> in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the <em>TaFUS3</em>-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of <em>TaFUS3</em> in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that<em> TaFUS3</em> plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.
文摘To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.
文摘The apoE cDNA was amplified from four months old fetus liver tissue mRNA by RT PCR. It was inserted into the TOPO ○R vector, whose sequence is the same as that reported. The right apoE cDNA was inserted into pQE30 vector to construct pQE30 apoE recombinant expressed plasmid, which was expressed as a histidine tag fusion protein in E.coli strain BL21(DE3) with higher level. The purified protein was gained by Ni NTA column. SDS PAGE showed that 34 kD protein was expressed. Western blot analysis showed a positive band at 34 kD.
