H. Phan, Nguyen Do Ngoc Nhi, L. Tien, Chu Thi Bich Phuong, Le Thi Kim Ngan, Phan Thị Phượng Trang, H. Nguyen
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引用次数: 3
摘要
在基础研究中,利用可调控的诱导型启动子表达少量蛋白质,研究其在细胞中的作用是十分必要的。pspace是一种以异丙基β- d -1-硫代半乳糖苷(IPTG)为诱导剂的枯草芽孢杆菌弱启动子。然而,携带该启动子的质粒(如pHCMC05)仍有一个缺点,即含有约200 bp的重复DNA片段,导致大肠杆菌结构不稳定,给克隆带来困难。方法:构建不携带重复序列的质粒,在大肠杆菌中研究质粒结构的稳定性,并测定β-半乳糖苷酶报告基因(bgaB)在枯草芽孢杆菌中的表达。结果:构建的质粒pHT2002在56代后结构稳定,而pHCMC05-bgaB在42代后结构不稳定,最终丢失。BgaB活性和Western-blot结果表明,iptg诱导启动子pspace控制的BgaB编码基因可以低水平表达。结论:研究表明,没有重复序列的新表达质粒在大肠杆菌中保持了结构稳定性,便于克隆。带pspace启动子的枯草芽孢杆菌表达质粒可以在IPTG存在下表达少量的异源蛋白。
Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter
Introduction: In basic research, it is essential to use an inducible promoter which can be controlled to express a small amount of protein for studying their roles in the cell. Pspac, a well-known weak promoter for Bacillus subtilis, uses isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. However, plasmids carrying this promoter such as pHCMC05 still have a disadvantage which harbors a repetitive DNA fragment of about 200 bp, resulting in structural instability in Escherichia coli, causing difficulty during cloning.
Methods: In this study, we constructed a plasmid that does not carry the repetitive sequences and investigated plasmid structural stability in E. coli, then measured the β-galactosidase reporter gene (bgaB) expression in B. subtilis.
Results: The constructed plasmid pHT2002 was stable over 56 generations while pHCMC05-bgaB was structurally instable and ultimately lost after 42 generations. BgaB activities and Western-blot indicated that BgaB-coding gene under control of IPTG-inducible promoter Pspac could be expressed at low levels.
Conclusion: The study demonstrated that the new expression plasmid without the repeated sequences retained its structural stability in E. coli facilitating the cloning step. The expression plasmid with Pspac promoter for B. subtilis could be used to express a modest amount of the heterologous protein in the presence of IPTG.