p16逆转录病毒载体的构建及其高滴度克隆的杂交筛选

Construction of p16 Retrovirus Vector and Identification of High-Titer Clones by the Nonradioactive vRNA Dot Blotting

构建多肿瘤抑制基因 p1 6( mts- 1 )的逆转录病毒载体 ,用一种简便的非放射性杂交方法 ,从多个包装细胞克隆中筛选出病毒滴度高的克隆 ,以提高病毒转导效率 .以 p1 6c DNA全长为目的基因 ,构建逆转录病毒载体 p LMSN,非脂质体转染试剂转染兼性包装细胞 PT67,G41 8筛选抗性克隆 ,扩增单个克隆后提取包装细胞上清的病毒 RNA( v RNA) ,以碱性磷酸酶直接标记的目的基因为探针作斑点杂文 ,化学发光自显影法定量测定 ,在短时间内从多个候选克隆中筛选出高产毒的克隆 .为 p1 6基因治疗的实验研究奠定了物质基础 .

As high frequent deletions and hypermethylation in promoter in human cancer cell, p16 is a major target in cancer gene therapy now. Retrovirus vecter pLMSN of p16 (mts 1)was constructed and a nonradioactive hybridizaton procedure was developed for obtain the high titer clones. Firstly the virus RNAs(vRNA)were extracted from the virus containing supernatant of packaging cell lines, then detected the vRNA by dot blotting with a alkaline phosphatase (AKP) labeled p16 cDNA probe and developed with the luminescent autophotography. The luminescent intensity was correlated with the virus titer determined by G418 colony screening method. The way is a repidly and simply preparation for latter research.