新型基因重组α-半乳糖苷酶工程菌菌种稳定性研究

Study of Heredity Stability of a Recombinant Plasmid pET28c-α-Galactosidase in Escherichia coli

观察脆弱类杆菌来源的新型重组α-半乳糖苷酶工程菌菌株的遗传稳定性。在有选择压力(Kan+)条件下,将重组α-半乳糖苷酶工程菌菌株在LB固体培养基上采用划线法连续传60代,每隔20代取样保存菌种,最后同时进行菌体形态、生长速度和抗生素抗性、平板传代及诱导过程中的质粒稳定性、限制酶切图谱、测序、表达量和酶活力检测。结果表明细菌形态、生长速度和抗生素抗性等与原始种子库无明显差异;LB固体培养基上传60代后质粒稳定性接近100%,但诱导过程中质粒易丢失。第20、40和60代提取质粒进行酶切检查,酶切图谱没有改变。DNA测序未见α-半乳糖苷酶基因变异。原代菌株及第20、40和60代菌株经诱导培养,其α-半乳糖苷酶表达水平、酶活力及菌体蛋白的SDS-PAGE图谱均无明显差异。

This study aimed to investigate the heredity stability of recombinant plasmid（pET28c-α-galactosidase）in host cell（BL21（DE3））.Samples were collected every 20 generations.Bacteria shape,growth rate,antibiotic resistance,plasmid stability,restriction map,sequencing,expressing level,and activity were analyzed.The results showed that all of the index was similar to the origin one,which indicated that recombinant stain,pET28c-α-galactosidase /BL21（DE3）had a high hereditable stability.