生长分化因子(BMP11)重组质粒在工程菌株中的稳定性研究
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摘要
为了研究重组质粒p ET28a-BMP11在基因工程菌E.coli BL21(DE3)中的遗传稳定性,将重组菌E.coli BL21(DE3)/p ET28a-BMP11液体转接传至100代,每隔20代取样观察菌落形态,检测菌体生长量、质粒保有率和目的蛋白表达水平,并对原代、第20代、第60代和第100代工程菌进行了XhoⅠ和NdeⅠ双酶切验证和目的基因测序。结果显示:各代菌生长量和菌体菌落形态与原代菌株无显著差异。在不含卡那霉素的条件下,传代进行到40代时,质粒保有率和蛋白质表达水平显著下降,第100代菌的质粒保有率仅为56%。在含有卡那霉素的条件下,第80代菌仍有较高的蛋白质表达水平,第100代菌酶切图谱和目的基因序列仍与原代保持一致,质粒保有率高达96%,说明利用重组大肠杆菌工业化生产BMP11是可行的,并且在培养基中添加卡那霉素能够让重组质粒在传代过程中保持稳定,为BMP11的大规模工业化生产和新型抗衰老食品的研发奠定了理论基础。
To investigate the hereditary stability of recombinant plasmid( p ET-BMP11) in host cell( Escherichiacoli BL21( DE3)).The recombinant E.coli was subcultured for 100 generation,samples were collected every 20 generations. Bacterial morphology,bacteria growth,plasmid stability,the target protein expressing level were analyzed. The XhoⅠ and NdeⅠ double enzyme validation and determination of target sequence were performed on the origin,20 th,60th,100 thgeneration strain. The results showed that the growth characteristics and the morphology of the cultures did not show differences among the generations,in the absence of kanamycin.The plasmid stability and protein expression of 40 thgeneration strain decreased significantly,and the plasmid retention rate of 100 thgenerations was only 56%. In the presence of kanamycin,the 80 th generation strain still had a high level of protein expression.The restriction enzyme map and target gene sequence of 100 thwere consistent with the original ones,and the rate of plasmid retention was up to 96%. These results showed that the use of recombinant E.coli to produce BMP11 was feasible,the addition of kanamycin in the medium kept the recombinant plasmid stable during passage,this study could provide a theoretical basis for the industrial production of BMP11 and the development of new anti-aging foods.
To investigate the hereditary stability of recombinant plasmid( p ET-BMP11) in host cell( Escherichiacoli BL21( DE3)).The recombinant E.coli was subcultured for 100 generation,samples were collected every 20 generations. Bacterial morphology,bacteria growth,plasmid stability,the target protein expressing level were analyzed. The XhoⅠ and NdeⅠ double enzyme validation and determination of target sequence were performed on the origin,20 th,60th,100 thgeneration strain. The results showed that the growth characteristics and the morphology of the cultures did not show differences among the generations,in the absence of kanamycin.The plasmid stability and protein expression of 40 thgeneration strain decreased significantly,and the plasmid retention rate of 100 thgenerations was only 56%. In the presence of kanamycin,the 80 th generation strain still had a high level of protein expression.The restriction enzyme map and target gene sequence of 100 thwere consistent with the original ones,and the rate of plasmid retention was up to 96%. These results showed that the use of recombinant E.coli to produce BMP11 was feasible,the addition of kanamycin in the medium kept the recombinant plasmid stable during passage,this study could provide a theoretical basis for the industrial production of BMP11 and the development of new anti-aging foods.
引文
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[3]Fadini G P,Menegazzo L,Bonora B M,et al.Effects of Age,Diabetes,and Vascular Disease on Growth Differentiation Factor11:First-in-Human Study[J].Diabetes Care,2015,38(8):118-119.
[4]Finkenzeller G,Stark G B,Strassburg S.Growth differentiation factor 11 supports migration and sprouting of endothelial progenitor cells[J].Journal of Surgical Research,2015,198(1):50-56.
[5]Wang Y,Wehlinghenricks M,Samengo G,et al.Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle-derived nitric oxide[J].Aging Cell,2015,14(4):678-688.
[6]沈巧艳,李娜,华进联.抗衰老的GDF11生物学特征与功能表现[J].中国生物化学与分子生物学报,2016,32(8):872-878.
[7]Yan Z,Jin S,Zhi W,et al.Growth differentiation factor 11 is a protective factor for osteoblastogenesis by targeting PPARgamma[J].Gene,2015,557(2):209-214.
[8]Sinha M,Jang Y C,Oh J,et al.Restoring systemic GDF11levels reverses age-related dysfunction in mouse skeletal muscle[J].Science,2014,344(6184):649-652.
[9]Katsimpardi L,Litterman N K,Schein P A,et al.Vascular and Neurogenic Rejuvenation of the Aging Mouse Brain by Young Systemic Factors[J].Science,2014,344(6184):630-634.
[10]Loffredo F S,Steinhauser M L,Jay S M,et al.Growth differentiation factor 11 is a circulating factor that reverses agerelated cardiac hypertrophy[J].Cell,2013,153(4):828-839.
[11]肖硕,洪华珠,彭建新.基因工程菌中重组质粒的稳定性研究进展[J].生物技术通报,2008,15(4):9-12.
[12]赵先萍.遗传工程中重组载体的构建及稳定性问题[J].农业开发与装备,2015,40(10):64.
[13]郭云萍,陶凌,李刚,等.重组人硫氧还蛋白工程菌生物学特性稳定性的检测[J].微生物学通报,2013,40(8):1457-1461.
[15]王宏华,凌红丽,侯竹美,等.鸡γ-干扰素基因重组质粒在工程菌株中的稳定性研究[J].微生物学通报,2008,35(7):1055-1058.
[16]翟成一,徐岩,聂尧,等.产普鲁兰酶重组大肠杆菌质粒稳定性的研究[J].工业微生物,2015,45(2):13-20.
[17]陈悦,李环,韦萍.产乙酰鸟氨酸脱乙酰基酶基因工程菌的构建及遗传稳定性研究[J].工业微生物,2008,38(2):23-27.