基因工程菌株BLG8900的遗传稳定性

Genetic Stability of Recombinant E. coli BLG8900 Strain

目的研究基因工程菌株BLG8900的遗传稳定性。方法在有选择压力(加氨苄西林)的条件下,测定pYC2质粒在大肠杆菌BLG8900株[含有质粒pYC2的BL21(DE3)]中的遗传稳定性。结果工程菌连续传10、25、50和100代后质粒具有良好的稳定性,而且经BamHⅠ/EcoRⅠ双酶切,酶切图谱相同。第100代菌株重组质粒的GnRH/TRS序列与原代菌株相同。原代与第10、25、50和100代菌株经IPTG诱导表达,GST-GnRH/TRS融合蛋白表达水平、菌体蛋白的SDS-PAGE图谱鉴定无明显差异。Western blot表明各代菌株表达产物均具有GnRH抗原特异性。结论质粒pYC2在大肠杆菌BL21(DE3)宿主菌中有较好的遗传稳定性。

Objective To study the genetic stability of recombinant E. coil BLG8900 strain. Methods Subculture recombinant E. coil BLG8900 strain,a BL21（DE3） strain containing plasmid pYC2,for different passages and test for the losing rate of plasmid. Results After subculture for 10,25,50 and 100 passages,the losing rate of plasmid pYC2 in recombinant E. coli BLG8900 strain were 1%, 1%, 2% and 2% respectively, and the restriction maps of bacteria were identical. The GnRH/TRS sequence of plasmid in recombinant E. coil bacteria passage 100 was identical to that in original recombinant E. coli. Both the expression levels and SDS-PAGE profiles of fusion protein GST-GnRH/TRS in recombinant E. coil BLG8900 strain of various passages showed no significant difference. The sequence of fusion gene in the BLG8900 strain of passage 100 was completely identical to that of passage 0. Western blot proved GnRH antigenic specificity of expressed product in BLG8900 strain of various passages. Conclusion The recombinant E. co/i BLG8900 strain containing plasmid pYC2 showed good genetic stability.