质粒pBV220-PTD-tCNTF转化BL21菌株感受态细胞的高效制备方法  

A high efficient preparation method of pBV220-PTD-tCNTF transformed into E.coli BL21 competent cells

作　　者：吴行伟[1,2] 刘泽源[2] 李前[3] 蒲韵竹[3] 任汝通[3] 许秀丽[3] 刘霏霏[2] 张琴[2] 吴媛[2] 曲恒燕[2] 

机构地区：[1]解放军总医院,北京100850 [2]军事医学科学院附属医院临床药理室,北京100071 [3]军事医学科学院毒物药物研究所,北京100850

出　　处：《中国药理学通报》2012年第4期584-587,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81001438)

摘　　要：目的比较几种常用大肠杆菌感受态制备方法,探讨影响pBV220-PTD-tCNTF转化大肠杆菌感受态细胞的因素并得出最优水平组合。方法采用4种制备感受态细胞的方法即CaCl2法,CaCl2-甘油法,TSSG法和INOUE法,比较4种方法制备的感受态细胞即时和-80℃保存30 d对质粒PBV220-PTD-tCNTF的转化效率;L8(27)正交设计研究Mg2+、INOUE重悬液、PEG、甘油和DMSO对pBV220-PTD-tCNTF转化大肠杆菌感受态的影响;对得出的最佳水平组合进行验证并对转化好的细胞进行测序验证。结果 CaCl2法,CaCl2-甘油法,TSSG法,INOUE法4种方法的转化率分别为:(5.43±0.72)×106、(7.45±0.65)×106、(9.71±1.81)×106、(8.52±1.22)×107;-80℃保存30 d的转化效率为:(6.20±0.31)×104、(6.57±0.97)×106、(9.76±1.31)×106、(1.63±0.019)×106;正交结果 Mg2+(A)、IN-OUE重悬液(B)、PEG(C)、甘油(D)和DMSO(E)各因素的P值分别为0.0357、0.0154、0.0677、0.2503和0.1749;最佳水平组合实施的转化率为(29.63±4.95)×107并经测序确定转入质粒的正确性。结论分析出各因素对pBV220-PTD-tCNTF转化感受态细胞转化率的影响程度,得出了其最佳水平组合A1B1C1D2E1,并得到了(29.63±4.95)×107的转化效率。Aim To compare several commonly used preparation methods of E.coli competent cells and to explore the influencing factors of pBV220-PTD-tCNTF transformed into E.coli cells to find an optimal level combination of the factors.Methods Four preparation methods were used for competent cells,namely,CaCl2,CaCl2-glycerol,TSSG and INOUE.Their transformation efficiencies of immediate and stored at-80℃ for 30 ds for pBV220-PTD-tCNTF after preparation were compared;factors of Mg2＋,INOUE re-suspension,PEG,glycerol and DMSO in the transformation of pBV220-PTD-tCNTF into E.coli were analysed by L8（27） orthogonal Design;the optimal levels combination of the factors were practised and the production was validated by cell sequencing.Results Methods of CaCl2,CaCl2-glycerol,TSSG,INOUE got their transformation efficiencies respectively as follows：（5.43±0.72）×106,（7.45±0.65）×106,（9.71±1.81）×106,（8.52±1.22）×107;transformation efficiencies of-80℃ saved for 30 ds：（6.20±0.31）×104;（6.57±0.97）×106;（9.76±1.31）×106;（1.63±0.019）×106;orthogonal results showed that factors of Mg2＋（A）,INOUE re-suspension（B）,PEG（C）,glycerol（D） and DMSO（E） got a P value of 0.0357,0.0154,0.0677,0.2503 and 0.1749 each;the Practice of the optimal levels combination of the factors got a transformation efficiency of（29.63±4.95）×107 and the cell sequencing showed its correctness of the plasmid transformed.Conclusion The various influence factors for the transformation efficiency of pBV220-PTD-tCNTF into the E.coli（BL21） are analysed,and its best levels combination of the factors A1B1C1D2E1 and a transformation efficiency of（29.63±4.95）×107 are attained.

关 键 词：感受态细胞 大肠杆菌 正交设计 转化效率 制备 PBV220 CNTF 

分 类 号：R318.02[医药卫生—生物医学工程] R378.21[医药卫生—基础医学]