重组大肠杆菌pET-28(a)/LF/BL21的传代稳定性  

Study on the generative stability of a recombinant Escherichia coli pET-28(a)/LF/ BL21

作　　者：李睿[1] 王革[1] 钱秋娟[1] 王秉翔[1] 

机构地区：[1]兰州生物制品研究所有限责任公司,兰州730046

出　　处：《微生物学免疫学进展》2012年第4期1-5,共5页Progress In Microbiology and Immunology

摘　　要：目的通过菌株传代方法观察携带致死因子(lf)基因的重组大肠杆菌pET-28(a)/LF/BL21的传代稳定性。方法将重组菌株pET-28(a)/LF/BL21在含有卡那霉素的LB培养基中连续传代至100代,比较第1、10、20、50、100代菌株的菌落形态、细菌形态、生化特性、质粒保有率、生长速度等的差异;取各代次菌株提取质粒DNA且分别进行双酶切及PCR鉴定、DNA测序;诱导表达各代次菌株,比较各代次菌株的重组rLF蛋白的表达水平及免疫反应性。结果各代次菌株菌落形态、生化特性、生长速度等方面与原始种子库无明显差异;在传至100代时的质粒保有率为76%;各代次菌株重组质粒电泳图谱、酶切图谱、PCR图谱未改变,未见lf基因变异;各代次菌株的总蛋白电泳图谱r、LF蛋白表达量r、LF蛋白的免疫反应性与原始种子库无明显差异。结论重组大肠杆菌pET-28(a)/LF/BL21具有较好的传代稳定性。Objective To observe the generative stability of a recombinant Escherichia coli pET-28 （ a）/LF/ BL21 strain carried/f gene through the subcultured method. Methods The pET-28（a）/LF/ BL21 had been subcultnred serialy to 100th generation. To observe the growth character and the morphology of the cultures among the generations;To observe the biochemistrical character and the plasmid＇ s consist percent of the pET-28 （ a）/LF/BL21 strain in different generations;To observe the chromatogram of the plasmid in different generations digested by BamHI and NcoI restriction enzymes and the PCR results of the recombinant plasmid ; To test the DNA sequences ; Comparing with the rLF expressed level and the anti- genicity in different generations. Results The growth charaeteristies ,the morphology of the cultures and the biochemistri-cal character did not showed difference among the generations. It was 76% cells containing plasmid after 100 generations. The chromatogram of the plasmid in different generations digested by BamHI and NcoI restriction enzymes and the PCR re-sult of the plasmid were the same. As well as DNA sequences showed no difference among generations. In addition to that or-igin strain and l0ts ,20th ,50＇s and 100ts generation strain had a similar rLF expressed level. The SDS-PAGE of the protein expressed and the antigenicity of the rLF in different generations were also the same. Conclusion Recombinant Escherichi- a coli pET-28（a）/LF/ BL21 has a good generative stability.

关 键 词：重组大肠杆菌pET-28(a)/LF/BL21 传代稳定性 

分 类 号：R378[医药卫生—病原生物学]