RNA解螺旋酶A表达系统的探究及其动力学研究

Investigation of expression system and dynamics properties of RNA helicase A

[目的]RNA解螺旋酶A（RNA helicase A,RHA）参与许多细胞生物学过程中,并能促进艾滋病病毒1型（HIV-1）等一些病毒的复制,本试验旨在探究温度、p H值对RHA的动力学影响。[方法]利用PCR扩增全长RHA基因,构建重组载体p ET-28a-RHA和p Cold-Ⅰ-RHA,分别转化至BL21 star（DE3）和BL21、Rosetta2菌株中,加IPTG分别在28℃和15℃中诱导表达。将全长RHA基因重组到p Fast Bac Dual载体中,然后将重组穿梭载体转化到含有杆状病毒基因组的DH10Bac感受态细胞中,通过转座作用,将RHA基因整合到杆状病毒基因组中。提取重组杆状病毒基因组（重组杆粒DNA）,将重组杆粒DNA转染到sf9细胞中,收集细胞,纯化蛋白,可以得到完整的RHA。建立体外解螺旋反应体系,改变温度、p H值,检测RHA活性的变化。[结果]完整RHA在BL21、Rosetta2中可微量可溶表达。sf9可以表达有活性的完整RHA。反应温度降低时,解螺旋速率变小,RHA活性降低。p H值在6.5-8.0范围内,随着p H值升高,RHA活性升高;当p H值大于8.0时,RHA活性降低。[结论]完整的RHA在BL21、Rosetta2中可微量表达;有3＇-tailed解旋极性的RHA活性受温度、p H影响较大。

[Objectives]RNA helicase A（ RHA） has been shown to participate in diverse cellular functions and promote replication of a number of viruses including human immunodeficiency virus 1（ HIV-1）. Its dynamics was studied in this paper. [Methods]The full-length RHA was amplified by PCR. DNA encoding full- length RHA was constructed in p ET-28 a and p Cold-Ⅰ plasmid,then transformed in BL21 Star（ DE3）,BL21 and Rosetta2 competent cells. The RHA gene was induced with IPTG at 28 ℃ or 15 ℃. The RHA gene was cloned into the p Fast Bac Dual donor plasmid and the p Fast Bac Dual construct was transformed into DH10 Bac to generate a recombinant bacmid. Then the recombinant bacmid DNA was transfected into the sf9 insect cells to generate a recombinant baculovirus. Recombinant protein was expressed by amplifying the baculovirus in infected sf9 insect cells. We established unwinding reaction system in vitro to detect the activity of RHA when the temperature and p H were changed. [Results]The soluble full-length RHA can＇t be detected in BL21 star（ DE3）,however it can be detected in BL21 and Rosetta2. RHA was purified and verified to be active. Decreasing the temperature repressed the activity of RHA. Lowering the p H value from 6. 5 to 8. 0 decreased the activity of RHA. [Conclusions]In this study,the full-length RHA gene was first verified to be expressed in bacteria expression system： BL21 and Rosetta2,although the expression level is low. RHA activity affected by p H and temperature was also first systematically studied.