重组人粒细胞-巨噬细胞集落刺激因子菌种稳定性研究

Studies on genetic stability of pBV220GM-CSF cultured in E.coli

为了解含重组质粒的大肠杆菌菌种连续传代及发酵罐培养中质粒的遗传稳定性,将GM-CSF菌种在含氨苄青霉素的琼脂平皿上连续划线传代培养,以及在不含安苄青霉素的培养基中进行发酵罐培养和42℃诱导表达.结果显示pBV220GM-CSF工程菌在氨苄青霉素选择压力下连续传代10,25,50和100次后质粒稳定、不丢失,而在不含氨苄青霉素的培养基中进行发酵罐大规模培养42℃诱导表达时,质粒稳定性下降,质粒易丢失,其表达量随诱导时间而变化.

The stabilities of pBV220GM-CSF were observed in continuous cultures and fermenter culture.The E.coli with pBV220GM-CSF was consecutively cultured on LB plates with ampicillin;fedbatch culture was made and GM-CSF was expressed by temperature inducing in ampicillin free media.The plasmids were stable in E.coli and the ability of expression and biological activity of GM-CSF was without obvious changes after 10,25,50 and 100 generational culture.But it was unstable at 42℃ inducing expression.This result showed that the plasmids of pBV220GM-CSF were stable in E.coli under antibiotics pressures.The suitable condition for large scale production should be explored.