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核技术、核医学、航空航天技术的迅猛发展给人类带来巨大福利的同时,也给人们带来不同程度的放射损伤甚至死亡。如何有效地预防和治疗放射损伤受到研究者的广泛关注,目前临床上治疗放射损伤多以化学合成小分子药物[1]、天然植物化合物[2]及对症支持治疗(成分输血、补液、补充电解质、应用抗生素等)为主,但疗效并不理想,人们迫切想找到高效、可行、低毒的辐射防护新方法。随着生物技术的不断发展,使辐射防护基因治疗成为可能,人们可以通过特定载体将辐射抗性基因定点整合到靶组织,提供额外的辐射防护以减少细胞损伤,提高细胞的增殖能力和生存率[3]。耐辐射奇球菌(Deinococcus radiodurans,DR)是目前地球上发现的最具辐射抗性的生物之一[4],可耐受超过12 kGy的电离辐射,是大多数脊椎动物的3000多倍[5]。pprM是DR特有的与辐射抗性密切相关的基因,缺陷菌株对电离辐射有较高的敏感性,其表达蛋白PprM可作为pprI介导DNA损伤响应的调节子[6-7],发挥着辐射抗性的作用。本研究构建重组绿色荧光真核表达载体pEGFP-C1-pprM,将DR高抗辐射基因pprM转入离体人肾上皮293T细胞中,并成功表达PprM蛋白,为后续实验研究pprM是否提高真核细胞的辐射抗性及其可能存在的作用机制奠定实验基础,为辐射防护与辐射损伤治疗开拓新途径。
耐辐射奇球菌pprM基因在真核细胞中的表达
Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells
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摘要:
目的扩增耐辐射奇球菌辐射抗性基因pprM,构建pEGFP-C1-pprM重组载体,转入293T细胞并表达PprM蛋白,为研究原核细胞辐射抗性基因pprM是否提高真核细胞的辐射抗性及其可能存在的作用机制奠定实验基础。 方法以无任何突变的pGEX-6p-1-pprM重组质粒为模版,设计引物PCR扩增pprM基因,利用琼脂糖凝胶DNA回收试剂盒纯化回收目的片段,EcoR Ⅰ、BamH Ⅰ双酶切回收的目的片段及质粒pEGFP-C1,将双酶切产物进行连接,连接产物转化至大肠杆菌JM109感受态细胞后涂布于含卡那霉素(Kan)抗性的Luria-Bertani(LB)固体培养基上进行筛选,所筛选的阳性克隆进一步用菌落PCR,EcoR Ⅰ、BamH Ⅰ双酶切及测序鉴定。通过Lipofectamine2000转染试剂将pEGFP-C1-pprM重组质粒转入293T细胞,倒置荧光显微镜观察绿色荧光融合蛋白的表达。裂解细胞抽提蛋白,Western blot进一步验证PprM蛋白的表达。 结果菌落PCR结果及双酶切结果显示,在400 bp左右处有一条明显的目的条带,测序结果显示碱基序列与原基因序列一致,载体构建成功。荧光拍照结果显示,pEGFP-C1-pprM重组质粒成功转染293T细胞并表达绿色荧光融合蛋白;Western blot结果显示在40×103处有融合蛋白表达。 结论笔者成功将所构pEGFP-C1-pprM重组质粒转入293T细胞,并表达相应蛋白,为后续实验研究原核基因pprM及其产物对真核细胞辐射抗性的影响奠定了良好的实验基础。 Abstract:ObjectiveTo enhance the radiation resistance of eukaryotic cells and to identify the mechanisms that underlie radiation resistance, the radioresistant pprM gene was amplified from Deinococcus radiodurans and used to construct a recombinant pEGFP-C1-pprM plasmid for the expression of PprM protein in 293T cells. MethodsThe pprM gene was amplified from pGEX-6p-1-pprM via PCR and purified from agarose gel with a DNA recovery kit. The purified PCR product was digested with EcoR Ⅰ and BamH Ⅰ and ligated into the pEGFP-C1 plasmid. Recombinant plasmids were transfected into competent JM109 cells, which were then cultured on LB solid medium that contained kanamycin. Positive clones were identified and characterized via bacterial colony PCR, restriction enzyme digestion, and sequencing analysis. Lipofectamine 2000 reagent was used to transfect pEGFP-C1-pprM plasmids into 293T cells. Green fluorescent fusion protein was observed via fluorescence microscopy and identified by Western blot. ResultsBacterial colony PCR and double digestion showed that the target band is approximately 400 bp in length. Sequencing results showed that the base sequence was identical to the original gene sequence, thus indicating the successful construction of the recombinant plasmid. Fluorescence photography results showed that pEGFP-C1-pprM plasmids were successfully transfected into 293T cells. Western blot results showed that fusion protein is approximately 40×103 in weight. ConclusionsThe pEGFP-C1-pprM plasmid was transfected into 293T cells, which then successfully expressed PprM protein. This study provides the foundation for future research on the pprM gene and the effects of its products on the radiation resistance of 293T cells. -
Key words:
- Deinococcus radiodurans /
- Radiation /
- pprM gene /
- 293T cells
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[1] Citrin D, Cotrim AP, Hyodo F, et al. Radioprotectors and mitigators of radiation-induced normal tissue injury[J]. Oncologist, 2010, 15(4):360-371. DOI:10.1634/theoncologist.2009-S104. [2] Szejk M, Kołodziejczyk-Czepas J, Żbikowska HM. Radioprotectors in radiotherapy-advances in the potential application of phytochemicals[J/OL]. Postepy Hig Med Dosw(Online), 2016, 30, 70(0):722-734[2016-12-20]. https://www.ncbi.nlm.nih.gov/pubmed/27356603. DOI:10.5604/17322693.1208039. [3] Everett WH, Curiet DT. Curiel gene therapy for radioprotection[J]. Cancer Gene Ther, 2015, 22(4):172-180. DOI:10.1667/RR14186.1. [4] Krisko A, Radman M. Biology of extreme radiation resistance:the way of Deinococcus radiodurans[J/OL]. Cold Spring Harb Perspect Biol, 2013, 5(7):a012765[2016-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685888/.DOI:10.1101/cshperspect.a012765. [5] Sai CH, Liao R, Chou B, et al. Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation[J]. Appl Environ Microbiol, 2015, 81(5):1754-1764. DOI:10.1128/AEM.03709-14. [6] Ohba H, Satoh K, Sghaier H, et al. Identification of pprM:a modulator of the pprI-dependent DNA damage response in Deinococcus radiodurans[J]. Extremophiles, 2009, 13(3):471-479. DOI:10.1007/s00792-009-0232-8. [7] Jeong SW, Seo HS, Kim MK, et al. pprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions[J]. J Microbiol, 2016, 54(6):426-431. DOI:10.1007/s12275-016-6175-8. [8] Mettler FA. Medical effects and risks of exposure to ionising radiation[J/OL]. J Radiol Prot, 2012, 32(1):N9-13[2016-12-20]. http://sci-hub.cc/10.1088/0952-4746/32/1/N9. DOI:10.1088/0952-4746/32/1/N9. [9] Li J, Li Q, Ma X, et al. Biosynthesis of gold nanoparticles by the extreme bacterium Deinococcus radiodurans and an evaluation of their antibacterial properties[J]. Int J Nanomedicine, 2016, 11:5931-5944. DOI:10.2147/IJN.S119618. [10] Passot FM, Nguyen HH, Dard-Dascot C, et al. Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans[J]. Mol Microbiol, 2015, 97(4):759-774. DOI:10.1111/mmi.13064. [11] Slade D, Radman M. Oxidative stress resistance in Deinococcus radiodurans[J]. Microbiol Mol Biol Rev, 2011, 75(1):133-191. DOI:10.1128/MMBR.00015-10. [12] Battista JR. Radiation resistance:The fragments that remain[J/OL]. Curr Biol, 2000, 10(5):R204-205[2016-12-20]. http://www.cell.com/current-biology/pdf/S0960-9822(00)00353-5.pdf.