Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm ...Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.展开更多
目的:利用5型腺病毒载体转染人T淋巴细胞,对其转染T淋巴细胞的效率以及细胞毒性进行分析。方法:选取T淋巴瘤Jurkat细胞及原代T细胞,腺病毒按感染复数(multiplicity of infection,MOI)为20、50、100、200和400对其转染,转染48 h后利用流...目的:利用5型腺病毒载体转染人T淋巴细胞,对其转染T淋巴细胞的效率以及细胞毒性进行分析。方法:选取T淋巴瘤Jurkat细胞及原代T细胞,腺病毒按感染复数(multiplicity of infection,MOI)为20、50、100、200和400对其转染,转染48 h后利用流式细胞术检测转染效率;选取腺病毒转染后的不同时点,利用碘化丙啶染色法分析转染对于细胞周期的影响;利用Annexin V/7-AAD染色法分析转染诱导细胞凋亡情况;利用台盼蓝染色计数法分析转染对活细胞数目的影响。结果:5型腺病毒载体转染T淋巴瘤的效率最高,转染效率随着MOI值的增大而增加;CD8+T细胞和CD4+T细胞的转染效率大致相同,T细胞经刺激活化后,CD8+T细胞的转染效率下降;病毒转染未导致明显细胞凋亡;病毒转染对细胞周期与活细胞数目没有显著影响。结论:5型腺病毒载体转染T细胞呈现较低细胞毒性。展开更多
文摘Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.
文摘目的:利用5型腺病毒载体转染人T淋巴细胞,对其转染T淋巴细胞的效率以及细胞毒性进行分析。方法:选取T淋巴瘤Jurkat细胞及原代T细胞,腺病毒按感染复数(multiplicity of infection,MOI)为20、50、100、200和400对其转染,转染48 h后利用流式细胞术检测转染效率;选取腺病毒转染后的不同时点,利用碘化丙啶染色法分析转染对于细胞周期的影响;利用Annexin V/7-AAD染色法分析转染诱导细胞凋亡情况;利用台盼蓝染色计数法分析转染对活细胞数目的影响。结果:5型腺病毒载体转染T淋巴瘤的效率最高,转染效率随着MOI值的增大而增加;CD8+T细胞和CD4+T细胞的转染效率大致相同,T细胞经刺激活化后,CD8+T细胞的转染效率下降;病毒转染未导致明显细胞凋亡;病毒转染对细胞周期与活细胞数目没有显著影响。结论:5型腺病毒载体转染T细胞呈现较低细胞毒性。
