重组TRAIL的纯化及其对非小细胞肺癌细胞株的杀伤活性

Purification of recombinant GST-TRAIL fusion protein and its sensitivity to non-small cell lung carcinoma cell lines

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 yield and purify the recombinant tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） active fragment, and to study the cytotoxic effects on non-small cell lung cancer （NSCLC） cell lines. Methods： The pGEX4T-1/TRAIL plasmid was transformed into BL21/DE3 bacteria. The IPTG was applied to induce the expression of TRAIL with GST tags （GST-TRAIL）. This was followed by purification using GST-BIND resin high-affinity chromatography And SDS-PAGE eleetrophoresis. The products were desalinated and concentrated, followed by quantification by using Bradford＇ s technique. The morphological characteristics of lung cancer cells were determined, and the cytotoxic activity of TRAIL on lung cancer cells was assayed by using lactate dehydrogenase （LDH） release technique. Results： about a total of 8.6mg purified TRAIL was derived. The IC50 was （2.33 ± 0.22 ） μg,/ml for A549, （50.0± 17.0 ） ng/ml for SPCA1, （ 872.0± 240.0 ） ng/ml for HLAMP, （ 427.0 ± 12.0 ） ng/ml for 95 D, （ 328.0 ± 28.0 ） ng/ml for PGCL3, （ 270.0 ± 33.0 ） ng/ml for 95 C and （178.0± 8.0）ng/ml for H460, respectively. Conclusion： The different lung cancer cell lines have different sensitivity to TRAIL, as evidenced by the order of sensitivity of A549, HLAMP, 95D, PGCL3, 95C, H460 and SPC-A1.