摘要
目的构建人源性DDX6多种突变体真核表达载体。方法以DDX6-WT为模板,利用同源重组技术构分别建出2个突变体,根据突变位置,分别命名为DDX6-EQ、DDX6-△C。结果经PCR、酶切、测序和Western Blot鉴定,构建出无解旋酶活性的DDX6-EQ和无ATP酶、解旋酶活性的DDX6-△C,蛋白分子量分别为54kD,34kD。结论成功构建2个突变体,为进一步研究DDX6在癌症进展过程中的调控作用奠定基础。
Objective To construct the different mutants of eukaryotic expression vector of human DDX6.Methods Using DDX6-WT as a template, two mutants were constructed by homologous recombination technique.According to the mutation position, the mutants were named DDX6-EQ and DDX6-△C, respectively. Results By PCR, enzyme digestion, DNA sequencing and Western blotting, DX6-EQ without helicase activity and DDX6-△C without both ATPase and helicase activity were constructed successfully. The molecular weights of protein were 54 and 34 kD, respectively. Conclusion Two DDX6 mutants were constructed successfully. It offers a foundation for the further study of the role of DDX6 in cancer progression.
引文
[1] Sloan KE,Bohnsack MT.Unravelling the Mechanisms of RNA Helicase ReguLation[J].Trends in Biochemical Sciences,2018,43(4):237-250.
[2]叶宏基,李少龙,安丽萍,等. DEAD-box RNA解旋酶与肿瘤关系的研究进展[J].世界科技研究与发展,2017,39(3):270-274.
[3] Cordin O,Banroques J,Tanner NK,et al.The DEAD-box protein family of RNA helicases[J].Gene 2006,367:17-37.
[4]Taniguchi K,Iwatsuki A,Sugito N,et al.Oncogene RNA helicase DDX6 promotes the process of c-Myc expression in gastric cancer cells[J].MolecuLar carcinogenesis,2018,57(5):579-589.
[5]文小红,张淑芳,张应爱.DEAD-box家族蛋白在癌症中的研究进展[J].中南大学学报(医学版),2017,42(11):1311-1315.
[6] Lin F,Wang R,Shen JJ,et al.Knockdown of RCK/p54 expression by RNAi inhibits proliferation of human colorectal cancer cells in vitro and in vivo[J].Cancer Biology&Therapy 2014,7(10):1669-1676.
[7] Yukihiro Akao,Masao Seto,Toshitada Takahashi,et al.MolecuLar cloning of the chromosomal breakpoint of a B-cell lymphoma with the t(11;14)(q23;q32)translocation[J].Cancer Res,1991,51(5):1574-1576.
[8]Yukihiro AKAO,Kenji Matsumoto,Kenji Ohguchi,et al.Human DEAD-box/RNA unwindase rck/p54 contributes to maintenance of cell growth by affecting cell cycle in cuLtured cells[J].International journal of oncology,2006,29(1):41-48.
[9]N. Kyle Tanner,Patrick Linde. DEx D/H Box RNA Helicases:From Generic Motorsto Specific Dissociation Functions.[J].MolecuLar Cell,2001,8(2):251-262.
[10]沈翠翠,黄培.DEAD box家族蛋白与大肠癌[J].世界华人消化杂志,2016,24(18):2811-2816.
[11] Rouya C,Siddiqui N,Morita M,et al.Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1[J].Rna 2014,20(9):1398-1409.
[12]Taniguchi K,Sugito N,Kumazaki M,et al.Positive feedback of DDX6/c-Myc/PTB1 reguLated by miR-124 contributes to maintenance of the Warburg effect in colon cancer cells[J].Biochimica et Biophysica Acta 2015,1852(9):1971-1980.
[13] Iio A,Takagi T,Miki K,et al.DDX6 post-transcriptionally down-reguLates miR-143/145 expression through host gene NCR143/145 in cancer cells[J].Biochimica et Biophysica Acta 2013,1829(10):1102-1110.
