鸡γ-干扰素基因重组质粒在工程菌株中的稳定性研究

Study on the Heredity Stability of a Recombinant Plasmid pET-ChIFN-γ in Escherichia coli

摘要:

本文通过传代的方法研究了鸡γ-干扰素基因的重组质粒pET-ChIFN-γ在工程菌CH1中的遗传稳定性。结果表明重组质粒在没有筛选压力下存在质粒不稳定性, 100代时质粒稳定性只有69％。适当的筛选压力可以显著降低质粒的遗传不稳定性, 100代时稳定性提高到95％。各代重组菌株在生长性能、质粒酶切图谱、蛋白表达等方面均保持一致, 未见明显差异。生物活性检测原代和100代均达到1.0×105 IU/mL以上。以上结果表明重组质粒pET-ChIFN-γ在工程菌株CH1中具有良好的遗传稳定性, 其质粒遗传不稳定性可由适当的筛选压力来控制。

Abstract:

A novel recombinant Escherichia coli BL21(DE3)/pET-ChIFN-γ was successfully constructed for high expression of chicken interferon-γ(ChIFN-γ), and the heredity stability of pET-ChIFN-γ was mainly focused on. It was segregationally unstable of the plasmid under no kanamycin selection pressure, only 69% cells containing plasmid after 100 generations. But it could be controlled by the supplementation of kanamy- cin pressure. The growth characteristics and the morphology of the cultures did not show differences among the generations. The chromatogram of the plasmids between origin strain and 100th generation strain di- gested by restriction enzymes were same, as well as ChIFN-γ expression level, biological activities showed no difference among the generations. It is indicated that recombinant strain CH1 has high heredity stability.