细胞因子转基因是目前肿瘤基因治疗研究最活跃的领域,其中IL-2基因转入肿瘤细胞的抗肿瘤效果最为肯定。IL-2基因转染后在肿瘤细胞中的表达水平与肿瘤细胞致瘤性丧失、抗亲代肿瘤能力呈正相关。In vivo 法基因治疗比ex vivo法更具有临床...细胞因子转基因是目前肿瘤基因治疗研究最活跃的领域,其中IL-2基因转入肿瘤细胞的抗肿瘤效果最为肯定。IL-2基因转染后在肿瘤细胞中的表达水平与肿瘤细胞致瘤性丧失、抗亲代肿瘤能力呈正相关。In vivo 法基因治疗比ex vivo法更具有临床前景,但目前常用的逆转录病毒载体体内易感染和整合于分裂期体细胞中,有引起与放化疗机理类似的剐作用的危险。因此in vivo法基因治疗要求目的基因在肿瘤细胞中高效特异表达。 1 材料和方法 1.1基因与载体:pSP65MIL-2质粒购自比利时Gent大学;pMNSM、pMNSM-TK-LacZ、Albe/p in Bluscripts质粒及HB101、JM109宿主菌,见文献。鸡β-actin cDNA由日本奈良医科大学Kuriyama博士赠送。 1.2动物与细胞株:BALB/c和C57BL/小鼠,3月龄,购于本校实验动物中心。PA317、NIH3T3TK-、MM45T.Li、MH134、BNLIME、B16、S180细胞株见文献,Psi2包装细胞由日本奈良医科大学Ku-riyama博士赠送。IL-2生物活性检测细胞CTLL-2引自波士顿大学。展开更多
Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant pl...Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant plasmid pLXSNPK was co transfected with packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (300 mg/L) for 2 weeks. Viral supernatants gaining stable cell clones were collected, the total RNA was extracted and the PK gene was amplified by RT PCR. It was shown that the PK gene had been inserted into the retroviral genome. The positive viral supernatant was collected to infect the interesting target cell MDBK. The infected MDBK cells were selected by G418 (800 mg/L) and analyzed by indirect immunofluorescence and SDS PAGE and Western blotting. It was confirmed that a MDBKPK cells expressing PK protein was obtained. It is useful to study the biological function of PrV PK gene in the process of PrV inducing culture cells apoptosis.展开更多
文摘Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant plasmid pLXSNPK was co transfected with packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (300 mg/L) for 2 weeks. Viral supernatants gaining stable cell clones were collected, the total RNA was extracted and the PK gene was amplified by RT PCR. It was shown that the PK gene had been inserted into the retroviral genome. The positive viral supernatant was collected to infect the interesting target cell MDBK. The infected MDBK cells were selected by G418 (800 mg/L) and analyzed by indirect immunofluorescence and SDS PAGE and Western blotting. It was confirmed that a MDBKPK cells expressing PK protein was obtained. It is useful to study the biological function of PrV PK gene in the process of PrV inducing culture cells apoptosis.
