Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design softwa...Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design software. The expected fragments about 170 bp, 287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion （Allium cepa L., Aggregatum group）, but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli. JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% to 90%,89.5% to 96.1%,and 91.6% to 96.3% in GLV, OYDV, and LYSV, respectively. The total RNA of 6.34 ug·uL^-1 from infected plants was diluted to a series of 10^-1 to 10^5 and the detection sensitivity of RT-PCR was 10^4 （about 4 ng）. Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.展开更多
Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasophar...Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma cell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting. The subcellular localization of NF-κB (p65) was detected by indirect immunofluorescence assay. The NF-κB transactivity was studied by transient transfection and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total IκBβ protein may have resulted from the initiation of an autoregulation loop after the activation of NF-κB. No change in the IκBβ level was detected. NF-κB (p65) was translocated from the cytoplasm to the nucleus following degradation of IκBα. After the introduction of the dominant-negative mutant of IκBα (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF-κB via phosphorylation and degradation of IκBα, but not IκBβ. The dominant-negative mutant of IκBα (Del 71) could completely inhibit both the nuclear translocation and transactivation of NF-κB induced by LMP1.展开更多
基金Supported by Department Education of Heilongjiang Province of China (10531146)
文摘Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus （GLV）, onion yellow dwarf virus （OYDV）, and leek yellow stripe virus （LYSV） by using PCR primer design software. The expected fragments about 170 bp, 287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion （Allium cepa L., Aggregatum group）, but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli. JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% to 90%,89.5% to 96.1%,and 91.6% to 96.3% in GLV, OYDV, and LYSV, respectively. The total RNA of 6.34 ug·uL^-1 from infected plants was diluted to a series of 10^-1 to 10^5 and the detection sensitivity of RT-PCR was 10^4 （about 4 ng）. Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.
基金ThisprojectwassupportedbytheStateKeyBasicResearchProgram FundamentalInvestigationonHumanCarcinogenesis (No G19980 5 12 0 1) +2 种基金theNationalScienceFundationforDistinguishedYoungScholars (No 395 2 0 2 2 ) andtheChinaMedicalBoardofNewYork Inc (N
文摘Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma cell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting. The subcellular localization of NF-κB (p65) was detected by indirect immunofluorescence assay. The NF-κB transactivity was studied by transient transfection and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total IκBβ protein may have resulted from the initiation of an autoregulation loop after the activation of NF-κB. No change in the IκBβ level was detected. NF-κB (p65) was translocated from the cytoplasm to the nucleus following degradation of IκBα. After the introduction of the dominant-negative mutant of IκBα (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF-κB via phosphorylation and degradation of IκBα, but not IκBβ. The dominant-negative mutant of IκBα (Del 71) could completely inhibit both the nuclear translocation and transactivation of NF-κB induced by LMP1.
