中国药理学通报2012，Vol.28Issue(4)：584-587,4.DOI:10.3969/j.issn.1001-1978.2012.04.033
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
摘要
Abstract
Aim To compare several commonly used preparation methods of E. coli competent cells and to explore the influencing factors of pBV220-PTD-lCNTF 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, CaCI2-glyceroI,TSSG and INOUE. Their transformation efficiencies of immediate and stored at - 801 for 30 ds for pBV220-PTD-tCNTF after preparation were compared; factors of Mg2- , INOUE re-suspension, PEG, glyeerol and DMSO in the transformation of pBV220-PTD-tCNTF into E. coli were analysed by Lg (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) xl06, (7.45 ±0.65) x 106, (9. 71 ± 1. 81) x 106, (8. 52 ± 1. 22) x 107; transformation efficien-cies of -80 saved for 30 ds: (6. 20 ±0. 31) xl04; (6.57± 0.97) xl06;(9.76 ±1.31) xl0';(1.63 ±0.019) xl06; orthogonal results showed that factors of Mg2 + (A) , INOUE re-sus-pension(B), PEG(C), glycerol(D) and DMSO(E) got a P value of 0. 0357,0. 0154,0. 0677,0. 2503 and 0. 1749 eachjthe Practice of the optimal levels combination of the factors got a transformation efficiency of (29.63 ±4. 95) x 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) xl07 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/CNTFKey words
competent cells/ E. coli/ orthogonal design/ transformation efficiency/ preparation/ PBV220/CNTF分类
医药卫生引用本文复制引用
吴行伟,曲恒燕,刘泽源,李前,蒲韵竹,任汝通,许秀丽,刘霏霏,张琴,吴嫒..质粒pBV220-PTD-tCNTF转化BL21菌株感受态细胞的高效制备方法[J].中国药理学通报,2012,28(4):584-587,4.基金项目
国家自然科学基金资助项目(No 81001438) （No 81001438）
