小鼠MIP1-α重组腺病毒载体的构建及其在肝癌细胞中的表达

Construction of adenoviral vector carrying murine MIP1-α and its expression in hepatocellular carcinoma

目的 :构建小鼠趋化因子 MIP1-α(m MIP1-α)的重组腺病毒载体并在小鼠肝癌细胞中表达 ,为肝癌基因治疗提供实验基础。 方法 :PCR扩增含有 m MIP1-α的质粒 ,将腺病毒骨架质粒 p Ad Easy- 1以及线性化的重组穿梭质粒 p Track- CMV-m MIP1-α共转化 BJ5 183受体菌并在其中发生同源重组 ,利用重组前后抗性的改变筛选出重组子 ,重组腺病毒质粒经过 2 93细胞的包装、扩增和纯化后 ,感染小鼠肝癌细胞株 Hepa1- 6 ,一步法提取细胞总 RNA,RT- PCR检测 m MIP1-α的 m RNA。琼脂糖凝胶打孔法体外观察感染上清中 m MIP1-α对小鼠脾细胞的趋化活性。 结果 :得到了携带 m MIP1-α的重组腺病毒 ,纯化后滴度为 4× 10 1 1 efu/ ml,在感染后的 Hepa1- 6细胞中检测到 m MIP1-α的表达 ,上清中 m MIP1-α的趋化指数为 6 .3。结论 :成功地构建了携带 m MIP1-α的重组腺病毒载体 ,为下一步应用于肝癌的基因治疗提供了基础。

Objective: To construct a recombinant adenoviral vector carrying murine MIP1 α(mMIP1 α) and to express it in hepatocellular carcinoma cell line. Methods: mMIP1 α was amplified from plasmid pBluescript/mMIP1 α, and homologous recombination was performed in BJ5183 bacteria by cotransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy 1. Recombinant plasmids were screened by alteration of antibiotic. The adenovirus vector was then packaged and amplified in 293 cells. The expression of mMIP1 α in infected murine Hepa1 6 cells was detected by RT PCR. The chemotaxis of mMIP1 α secreted in the supernatants to mouse spleen cells was observed on agarose gel. Results: The recombinant adenoviral vector carrying mMIP1 α was constructed, whose titer was 4×10 11 efu/ml after purification, and the mMIP1 α mRNA was detected in the infected murine Hepa1 6 cells. Bioactive mMIP1 α was detected in the supernatants. Conclusion: The recombinant adenoviral vector carrying mMIP1 α is successfully constructed, paving the way for further application in cancer gene therapy.