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目的:克隆人截短型凋亡诱导因子(AIF)cDNA序列,并构建早期生长反应因子1(Egr1)介导的重组表达载体pcDNA3.1-Egr1-AIFΔ1-480(pEgr1-AIFΔ1-480),观察其在人乳腺癌MCF-7细胞中的辐射诱导表达规律。方法:以人白血病Jurkat细胞mRNA为模板,RT-PCR法扩增获得人AIFΔ1-480,与pMD18T载体连接后行全自动测序,限制性内切酶切取pMD19T-Egr1中Egr1片段,并利用基因重组技术构建Egr1启动子介导的人AIFΔ1-480表达质粒pEgr1-AIFΔ1-480。实验分为对照组、pcDNA3.1组、pAIFΔ1-480组和pEgr1-AIFΔ1-480组。各组质粒分别转染MCF-7细胞,Western blotting法检测AIF及AIFΔ1-480蛋白的辐射诱导表达时程-效应(2.0Gy照射后0、2、4、12、24和48h)和剂量-效应(0、0.2、0.5、1.0、2.0、5.0Gy照射后24h)规律。结果:经测序证实,RT-PCR获得的人AIFΔ1-480基因与预期一致,pEgr1-AIFΔ1-480经PCR和酶切鉴定完全正确;各组MCF-7细胞经2.0Gy照射后0～48h,AIF蛋白在各组中均有表达,从4h开始显著增加,4、12、24和48h各组AIF表达与0h组比较差异有统计学意义(P<0.05),48h达到最大;而在pAIFΔ1-480和pEgr1-AIFΔ1-480组,AIFΔ1-480从2h开始表达,4、12、28和48h各组AIFΔ1-480表达与2h比较差异有统计学意义(P<0.05),24h达到峰值;而且24和48h时pEgr1-AIFΔ1-480组AIFΔ1-480表达较pAIFΔ1-480组显著增加(P<0.05)。各组MCF-7细胞经0～5.0Gy照射后24h,各组均有AIF蛋白表达,而AIFΔ1-480只在pAIFΔ1-480和pEgr1-AIFΔ1-480组表达,二者均随着剂量增加而增加,0.2、0.5、1.0、2.0和5.0Gy照射后各组AIF表达与0Gy比较差异有统计学意义(P<0.05),在5.0Gy照射时达到最大,相同照射剂量pEgr1-AIFΔ1-480组AIFΔ1-480表达高于pAIFΔ1-480组。结论:成功构建Egr1介导的人截短型AIF重组表达载体pEgr1-AIFΔ1-480,AIF和AIFΔ1-480蛋白在MCF-7细胞中的表达随照射时间延长和照射剂量增加而增加。
OBJECTIVE: To clone human truncated AIF cDNA sequence and construct recombinant plasmid pcDNA3.1-Egr1-AIFΔ1-480 (pEgr1-AIFΔ1-480) mediated by Egr1. To observe its radiation-induced expression in human breast cancer MCF-7 cells. Methods: Human AIFΔ1-480 was amplified by RT-PCR from human leukemia Jurkat cell mRNA. After being ligated with pMD18T vector, the full-automatic sequencing was performed. The Egr1 fragment of pMD19T-Egr1 was excised by restriction endonuclease. Recombinant technology to construct Egr1 promoter-mediated human AIFΔ1-480 expression plasmid pEgr1-AIFΔ1-480. The experiment was divided into control group, pcDNA3.1 group, pAIFΔ1-480 group and pEgr1-AIFΔ1-480 group. Each group of plasmids were transfected into MCF-7 cells respectively. The time-response (0, 2, 4, 12, 24 and 48 h after AAV exposure) and dose-response (0,0.2,0.5,1.0,2.0,5.0Gy 24h after irradiation) law. Results: The sequencing confirmed that the human AIFΔ1-480 gene obtained by RT-PCR was consistent with the expected, pEgr1-AIFΔ1-480 was completely correct by PCR and restriction enzyme digestion. MCF-7 cells in each group were exposed to 2.0Gy 0-48h, AIF The protein expression in each group was significantly increased from 4h, the expression of AIF in 4, 12, 24 and 48h groups was significantly different from that in 0h group (P <0.05), and reached the maximum at 48h; while in pAIFΔ1-480 And pEgr1-AIFΔ1-480 group, AIFΔ1-480 expression began at 2h, the expression of AIFΔ1-480 in each group at 4, 12, 28 and 48h was significantly different from that in 2h (P <0.05), and peaked at 24 h At 48h, the expression of AIFΔ1-480 in pEgr1-AIFΔ1-480 group was significantly higher than that in pAIFΔ1-480 group (P <0.05). The expression of AIF protein was detected in all groups at 24 hours after 0 ~ 5.0Gy irradiation, while AIFΔ1-480 was only expressed in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups, both of which increased with increasing dose (P <0.05). The AIFΔ1-AIFΔ1-480 group at the same irradiation dose had the highest AIFΔ1-AIFΔ1-480 expression at the same irradiation dose (P <0.05) 480 expression was higher than pAIFΔ1-480 group. CONCLUSION: The expression of pEgr1-AIFΔ1-480, AIF and AIFΔ1-480, which were successfully constructed in Egr1-mediated truncated AIF expression vector in MCF-7 cells, increased with time and dose of irradiation.