耐辐射奇球菌pprM与pprI基因修饰对真核293T细胞辐射抗性 的影响
摘要：
研究背景与目的：耐辐射奇球菌（Deinococcus radiodurans, DR）是已发现物种中电离辐射耐受性最强的生物之一，可耐受超过15kGy的电离辐射，其辐射耐受剂量是大多数脊椎动物的3000多倍。pprI基因是DR中参与DNA损伤修复的一个关键基因，是辐射响应调控的总开关。pprM基因是DR中特有的辐射响应基因，pprM基因缺失突变株对γ辐照抗性明显降低。本实验旨在研究DR辐射响应基因pprI、pprM的表达对293T细胞辐射抗性的影响，为辐射损伤提供新思路。
研究方法：1、以实验室前期构建的pGEX-6p-1-pprM为模板，PCR 扩增pprM基因，构建pEGFP-C1-pprM绿荧光真核表达载体。
2、将pEGFP-C1-pprM、pDsRed1-N1-flag-pprI（实验室前期构建）分别单独转入及两质粒共转入293T细胞。倒置荧光显微镜及激光共聚焦显微镜观察红绿荧光融合蛋白的表达及在细胞内的共定位。Western blot进一步验证pprI、pprM基因融合蛋白的表达。
3、MTT法检测293T细胞的生存率，荧光探针DCFH-DA检测细胞内活性氧（ROS）含量，WST-1法测总
超氧化物歧化酶（SOD）活力，TBA微板法检测丙二醛（MDA）含量及还原型谷胱甘肽（GSH）活力。
研究结果：1、测序鉴定及Blast比对分析结果显示pEGFP-C1-pprM
I
构建载体序列与模板基因碱基序列一致，载体构建成功。
2、倒置荧光显微镜下观察到红绿荧光蛋白的表达量较高，激光共聚焦显微镜下观察到PprI、PprM融合蛋白在293T细胞细胞核与细胞质中存在共定位现象。Western blot结果显示，在约40kD及65kD处有明显条带，分别为pprM、pprI基因所表达的融合蛋白。
3、与未转染组相比，空质粒转染组细胞无明显差异，pprM、pprI 单基因转染组及pprM+pprI双基因转染组细胞辐照后生存率、总SOD 活力及GSH活力明显增高（P <0.05），细胞内ROS及MDA含量明显降低（P <0.05），且pprM+pprI共转染组比两基因单转染组效果更为明显，差异有统计学意义。 结论：1、原核DR-pprM、pprI基因能够在真核293T细胞中表达，且PprM、PprI蛋白在细胞内存在共定位，可能存在相互作用。
脊灰野病毒
2、DR-pprM、pprI及pprM+pprI基因在293T细胞中的表达能够提高辐射损伤后细胞的增殖能力和抗氧化能力。
3、DR-pprM+pprI双基因转染效果比两基因单转染的细胞增殖能力和抗氧化能力提升效果更为明显，pprM与pprI基因间可能存在协同抗辐射作用。
关键词：耐辐射奇球菌；pprM；pprI；抗辐射；抗氧化；基因转染；293T细胞
拉大剧II
EFFECTS ON THE RADIATION RESISTANCE OF EUKARYOTIC 293T CELLS TRANSFECTED WITH DEINOCOCCUS RADIODURANS pprM AND pprI GENEpp视频加速器
余热锅炉Yang Qi (BioloGy)
Directed by Professor He Shu-Ya
Abstract:
Background and Objective: Deinococcus radiodurans (DR) is one of the most resistant organisms on
the earth. It could survive from ionizing radiation of more than 15kGy, which is 3000 times higher than most vertebrates. pprI is a key gene involved in DNA repair after ionizing radiation and it is a general switch responsible for extreme radioresistance of DR. pprM is a specific radiation response gene in DR, the deletion mutant of pprM is more sensitive toγ-radiation than the wild .The aims of this study were to investigate the effects on the radiation resistance of 293T cells transfected with pprM and pprI gene and provide a new idea for radiation injury treatment.
Methods: 1. pprM gene was amplified from pGEX-6p-1-pprM to construct pEGFP-C1-pprM plasmid .
2. pEGFP-C1-pprM、pDsRed1 - N1 - flag - pprI plasmids were transfected into 293T cells. The fusion proteins and their locations were observed by inverted fluorescence microscope and laser scanning confocal
III
microscope. The proteins were identified by Western blot.
3. The survival rates of 293T cells were determined by MTT. Using fluorescent probe DCFH-DA to detect reactive oxygen species (ROS) content in cells. WST-1 method and microplate test were used
to detect superoxide dismutase (SOD) activity and malondialdehyde (MDA)content,glutathione (GSH) activity , respectively.
Results:1. The sequence and Blast comparison results showed that the constructed vector sequence was consistent with the template base. The pEGFP-C1-pprM recombinant plasmid was constructed successfully.
2. The observed proteins of red andgreen fluorescent indicated that PprI and PprM were successfully expressed in 293T cells.The results observed by laser scanning confocal microscope showed that PprI and PprM have co-localizations in 293T cells. Western blot results showed that PprI and PprM fusion proteins were found at about 65kD and 40kD .
3. There was no significant difference between non-transfectedgroup and thegroup transfected with empty plasmid. However, the survival rate、SOD andgSH activity ofgroups transfected with pprM、pprI and pprM + pprI were significantly increased (P <0.05). The contents of ROS and MDA were significantly decreased (P <0.05).
Conclusion:1.The PprI and PprM proteins are successfully expressed in
293T cells and they have co-localizations in cells.
IV
2.The successful expression of DR-pprM, pprI and pprM + pprI genes in 293T cells can enhance their survival rate and antioxidant capacity.脂肽
异丙酚3.DR-pprM + pprI transfectedgroup effects is more notablely than pprM and pprI transfected respectively, there may be synergistic anti-radiation effects between pprM and pprI gene.
Keywords: Deinococcus radiodurans; pprM; pprI; radiation resistance; antioxidant;gene transfected; 293T cells
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