c-myb反义RNA重组载体构建及其包装细胞株的建立

Construction of c-myb Antisense RNA Recombinant Retroviral Vector and Establishment of Its Packaging Cell Line PA317

【目的】构建反义c myb重组逆转录病毒载体并建立其包装细胞株。【方法】采用RT PCR方法 ,获取目的基因 ,通过TA克隆方法克隆入 pUC19,再反向亚克隆入逆转录病毒载体 pDOR中。采用DOTAP法将重组逆转录病毒载体转入包装细胞 ,以NIH3T3细胞为靶细胞测定产病毒滴度。【结果】序列测定结果与GenBank中序列一致。c myb基因片段已定向克隆入 pDOR。细胞转染表明 ,已形成稳定的包装细胞株PA317/pDOR myb ,产病毒滴度达 5 2× 10 4 ～ 9 5× 10 4 CFU/mL。【结论】成功构建了含有反义c myb的重组逆转录病毒质粒 。

To construct the c myb antisense RNA recombinant retroviral vector and its packaging cell line. The segment of c myb gene was cloned into pUC19 with TA cloning method after amplication by RT PCR, and then was subcloned into retroviral vector pDOR. The recombinant retroviral vector named pDOR myb was transfected into retroviral package cell line PA317 after selection with G418. Sequencing data indicated that the c myb gene was exactly identical to the sequence in the GenBank. The segment of c myb gene was inserted directionally into pDOR. Resistant colonies were obtained and the titers of pDOR myb were 5 2×10 4～9 5×10 4 CFU/mL. [Conclusion] The recombinant retroviral vector containing c myb gene is successfully constructed and its packaging cell line PA317/pDOR myb was established.