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目的:表达、纯化重组肿瘤坏死因子相关凋亡诱导配体(TRAIL)的活性片段,并观察其对非小细胞肺癌细胞株的杀伤活性。方法:利用已有的pGEx-4T-l/TRAIL重组质粒转化BL21/DE3菌;异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达谷胱甘肽转移酶(GST)标签的TRAIL,用GST-BIND树脂亲和层析柱纯化;SDS-PAGE电泳分析纯化结果;蛋白质超滤除盐、浓缩TRAIL;Bradford法定量。形态学观察肺癌细胞;乳酸脱氢酶(LDH)释放法确定TRAIL对肺癌细胞的杀伤活性。结果:共获得8.6 mg纯化TRAIL,其对A549、SPC-Al、HLAMP、95D、PGCL3、95C、H460等7株细胞株的IC50值分别为(2.33±0.22)μg/mL、(50.0±17.0)、(872.0±241.0)、(427.0±12.0)、(328.0±28.0)、(270.0±33.0)、(178.0±8.0)ng/mL。结论:相同组织类型的不同肺癌细胞株对重组TRAIL的敏感度不同,从低到高依次为A549、HLAMP、95D、PGCL3、95C、H460和SPCA1。
OBJECTIVE: To express and purify the active fragments of recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to observe their cytotoxicity against non-small cell lung cancer cell lines. Methods: BL21 / DE3 was transformed with pGEx-4T-1 / TRAIL recombinant plasmids. IPTG induced glutathione transferase (GST) tag TRAIL, purified by GST-BIND resin affinity chromatography; SDS-PAGE electrophoresis analysis; protein ultrafiltration desalination; TRAIL concentration; Bradford assay. Morphological observation of lung cancer cells; Lactate dehydrogenase (LDH) release assay to determine the killing activity of TRAIL on lung cancer cells. Results: A total of 8.6 mg of purified TRAIL was obtained. The IC50 values of the 7 cell lines A549, SPC-Al, HLAMP, 95D, PGCL3, 95C and H460 were (2.33 ± 0.22) μg / mL and (50.0 ± 17.0) , (872.0 ± 241.0), (427.0 ± 12.0), (328.0 ± 28.0), (270.0 ± 33.0) and (178.0 ± 8.0) ng / mL. CONCLUSION: The sensitivity of different lung cancer cell lines of the same tissue type to recombinant TRAIL is different from A549, HLAMP, 95D, PGCL3, 95C, H460 and SPCA1 from low to high.