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目的:利用逆转录病毒载体pBaba-puro构建携带ROS1基因及其CD74-ROS1融合基因的重组载体pBaba-puro-ROS1,pBaba-puro-CD74-ROS1。方法:设计与合成引物,提取组织标本RNA,反转录和PCR扩增,经BamHI和TaqI双酶切,琼脂糖凝胶电泳,切胶回收进行连接转化,并再次酶切鉴定,测序分析。结果:成功构建携带ROS1基因及其CD74-ROS1融合基因的重组载体pBaba-puro-ROS1,pBaba-puro-CD74-ROS1,并通过双酶切与测序鉴定。结论:利用逆转录病毒载体基因重组技术能够成功构建出携带相应基因的逆转录病毒,可用于后续研究。
OBJECTIVE: To construct the recombinant vectors pBaba-puro-ROS1 and pBaba-puro-CD74-ROS1 carrying ROS1 gene and CD74-ROS1 fusion gene by retroviral vector pBaba-puro. Methods: The primers were designed and synthesized, RNA of tissues was extracted, reverse transcription and PCR amplification were carried out. After digested by BamHI and TaqI, agarose gel electrophoresis and gel excision were used for transformation and connection, and were digested and identified again and sequenced and analyzed. Results: The recombinant vectors pBaba-puro-ROS1 and pBaba-puro-CD74-ROS1 carrying the ROS1 gene and its CD74-ROS1 fusion gene were successfully constructed and identified by double enzyme digestion and sequencing. Conclusion: The retrovirus carrying the corresponding gene can be successfully constructed by retroviral vector gene recombination technology and can be used in subsequent studies.