【摘要】 目的探讨卡介苗(BCG)电转化中影响转化效率的因素。方法利用电转化仪将穿梭质粒pYL-hB7-2转入BCG中,考察其影响因素并优化转化条件。转化子利用卡那霉素抗性筛选和聚合酶链反应(PCR)及酶链免疫吸附试验(ELISA)鉴定。结果重组BCG获得较高的转化效率,最高可达8/9。外加电场强度和质粒浓度是决定电转化效率的关键因素,感受态细胞的状态,如生长期和冰浴时间均可影响转化效率。结论成功解决了基因重组BCG构建过程中电转化的问题。更多还原

【Abstract】 Objective To study the method of electro-transformation of BCG(Bacille Calmette-Guerin)and the factors that affect transformation efficiency. To investigate the expression of IFN-α by PBMCs after stimulation with recombinant bacille Calmette-Guérin (BCG) expressing human B7-2, and to study the antitumor effect of BCG activated killer cells(BAK). Methods Electroporation was performed on gene pulser with E.coli-Mycobacteria shuttle vector pYL-hB7-2 accordingly. Transformants were screened with kanamycin resistance and confirmed by PCR amplification of DNA fragment and ELISA. Results Electro-transformation of BCG was successfully accomplished with a high efficiency of 8/9. The hybrid plasmids maintained genetically stable in hosts. Primary results showed that variables that affected transformation efficiency were voltage gradient, concentration of plasmids DNA and the conditions of competent cell, such as growth stage and incubation time on ice before washing. Conclusion This study has successfully solved the problem of BCG transformation in the research of genetic engineering recombinant BCG vaccines.更多还原