海岸带生物学与生物资源保护实验室知识库

南京农业大学江苏省海洋生物学重点实验室;中国科学院烟台海岸带研究所;  

Genetic stability is essential in the production practice of interest product.Therefore,in order to determine the genetic stability of the genetic strain,plasmid stability was identified through methods of determination of cell growth,plate streaking,enzyme cutting of plasmid DNA,product identification.The experiment shows the following results： the plasmid loss rate of strain His-β was 6% for 5 generation,8% for 10 generation,9% for 15 generation,15% for 20 generation during the subculture on the solid plate with no antibiotics;the size and shape of colony was essentially the same after the strain was transferred of culture on the solid plate for 20 generations;recombinant plasmids did not change before and after subculture through the identification of BamHⅠand HindⅢenzyme cutting;His-β protein in primary and 5,10,15 and 20 generations can be expressed and showed no significant difference after induction.The results showed that the plasmid had structural stability and segregational instability.

工程菌株的遗传稳定性在目的产物的生产应用中至关重要。为了确定工程菌株的遗传稳定性,通过对重组别藻蓝蛋白β亚基(His-βAPC)生产菌株E.coliJM109(DE23)/pET28α-βAPC进行了菌体生长量的测定,平板划线,质粒酶切,产物鉴定等研究,检验了该工程菌质粒的稳定性。通过实验得到如下结果:该工程菌株在没有抗生素选择压力下传代,质粒丢失率为5代6%,10代8%,15代9%,20代15%;该菌株经固体平板连续划线传代20次后,菌落大小和形态基本不变;质粒经BamHⅠ和HindⅢ酶切后进行琼脂糖凝胶电泳结果显示该菌株携带的重组质粒目的片段在传代前后没有发生变化;经诱导培养后,His-βAPC在原代和第5、10、15和20代宿主菌中都可以表达,表达量没有明显差别,且表达产物在SDS-PAGE中的带型基本一致。以上结果表明,该工程菌质粒具有结构稳定性和分裂不稳定性。