重组α-N-乙酰半乳糖胺酶工程菌菌种稳定性研究

Study of heredity stability of a recombinant plasmid pET22b-a-NAGA in Escherichia coli

目的探讨重组α-N-乙酰半乳糖胺酶工程菌菌种的遗传稳定性。方法在有选择压力(AMP+)条件下,将重组a-N-乙酰半乳糖胺酶工程菌菌株在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、质粒稳定性、限制酶切图谱、测序、表达量和酶活力及Westernblotting检测。结果第0、20、40、60代菌的菌体形态、生长速度和抗生素抗性等与原代菌株无明显差异;LB固体培养基上传60代后质粒稳定性接近100%;DNA测序未见α-N-乙酰半乳糖胺酶基因变异;SDS-PAGE图谱显示,α-NAGA表达水平无明显差别。Western blotting检测显示重组α-NAGA能与抗His的单抗特异结合。结论α-N-乙酰半乳糖胺酶重组质粒pET22b-α-NAGA在工程菌株中具有良好的遗传稳定性。

Objective To investigate the heredity stability of recombinant plasmid （pET22b-a-NAGA）in host cell [ BL21 （DE3） ]. Methods Samples were collected every 20 generation. Bacteria shape, growth rate, antibiotic resistance, plasmid stability,restriction map,sequencing,expressing level,activity were analyzed. Results The passage 20^th ,40^th and 60^th Bacteria morphology, growth rate and antibiotic resistance were not evidently different from the origin ones＇. The stabili- ty of the plasmid was almost 100% after 60 passages in LB solid medium. There was no variation in a-NAGA after DNA sequencing,no evident difference in a-NAGA expressing level after SDS-PAGE, and the results of Western Blotting showed that the recombinant a-NAGA can bind specifically with anti-his monoclonal antibody. Conclusion The study results indicates that the recombinant plasmid pET22b-a-NAGA has high heredity stability in BI21 （DE3） strain.