重组精氨酸脱亚胺酶工程菌的遗传稳定性

Genetic Stability of Recombinant Arginine Deiminase Engineered Strain

考察重组精氨酸脱亚胺酶工程菌E.coli BL21/p ET27b-r ADI的遗传稳定性。在有抗生素(卡那霉素)选择压力的条件下,采用SB平板划线连续传代50代,每10代取样保藏菌种,并进行菌落形态、质粒稳定性、限制性内切酶酶切图谱、测序、蛋白表达量及重组蛋白活性检测。结果表明,10、20、30、40和50代工程菌的菌落形态及抗生素抗性与原始工程菌无显著差异,质粒的酶切图谱一致,各代质粒稳定,DNA测序未见基因突变或丢失;经异丙基-?-D-硫代半乳糖苷诱导后,原始及传50代工程菌的重组精氨酸脱亚胺酶表达水平和蛋白质活性无显著差异。

The genetic stability of arginine deiminase （ADI） engineering strain, E. coli BL21/pET27b- rADI, was investigated. The engineering strain was passaged under antibiotic selective pressure （kanamycin＋） on SB agar plates for 50 generations. Every ten generations were preserved and their colony morphology, plasmid stability, restriction map, ADI gene sequencing, expressing level and activity of the recombinant protein were analyzed. The results showed that the engineering strain maintained the typical characteristics after continuous passages, without significant difference in terms of growth rate compared with the parental strain. The restriction map of plasmids was correct after 50 generations. There were no DNA mutations or missing. After isopropyl p-D-1-thiogalactopyranoside induction, SDS- PAGE of cell extractions showed no significant differences in the recombinant arginine deiminase expression levels.