多肽pd20与肿瘤坏死因子α融合蛋白的纯化及生物学活性鉴定

The Purification and Identification of the Biological Activity of Fused Protein pd20-TNF-α

背景多肽pd20是具有胃癌肝转移导向性的新多肽分子,为研究其是否具有携带抗癌药物或抑癌基因靶向性治疗胃癌肝转移的作用,本研究将其与肿瘤坏死因子α(TNF-α)进行基因融合,构建原核表达载体,对重组融合蛋白进行诱导表达。目的将pd20-TNF-α融合蛋白进行蛋白纯化,并对纯化后蛋白进行鉴定和生物学活性检测。方法用Ni-NTA柱法纯化pd20-TNF-α融合蛋白并进行Western blotting检测;采用L929细胞毒法进行pd20-TNF-α融合蛋白的生物学活性检测;用流式细胞术检测不同浓度融合蛋白作用于胃癌肝高转移潜能细胞XGC9811-L的早期凋亡率,此实验分为5组:pd20-TNF-α100.00、25.00、6.25 U/ml分别为A、B、C组,不加融合蛋白的为D组,流式细胞凋亡的KB组为E组;细胞侵袭实验观察融合蛋白对胃癌肝高转移潜能细胞XGC9811-L转移能力的影响,此实验分为4组:pd20-TNF-α组、TNF-α组、XGC9811-L组和对照肽组。结果通过Ni-NTA柱纯化,在咪唑浓度为200 mmol/L时获得纯化的目标pd20-TNF-α融合蛋白,蛋白纯度可达92%;纯化后的蛋白具有与抗TNF-α单抗结合的能力。L929细胞毒法显示:pd20-TNF-α融合蛋白对L929细胞有明显的杀伤力,杀伤活性可达7.6×106U/ml。凋亡实验显示:A^E 5组细胞早期凋亡率间有差异〔(9.04±0.08)%、(6.96±1.21)%、(5.99±0.02)%、(0.73±0.02)%、(0.01±0.00)%;F=2.57,P<0.05〕。细胞侵袭实验显示:pd20-TNF-α组、TNF-α组、XGC9811-L组、对照肽组单位时间内的穿膜细胞数有差异〔(26.5±5.9)、(36.0±3.2)、(63.5±5.0)、(64.0±6.8)个;F=49.87,P<0.01〕。结论本研究成功纯化了pd20-TNF-α融合蛋白,并证实该蛋白有明显的生物学活性,具有促进胃癌细胞早期凋亡和抑制胃癌细胞侵袭的能力。

Background pd20 polypeptide is a kind of new small molecule polypeptide, which is hepatic metastasis from gastric cancer oriented. In order to verify whether pd20 has the ability to treat hepatic metastasis from gastric cancer when car- rying antitumor drugs or tumor suppressor gene, pd20 gene was fused with TNF-α gene to construct a prokaryotic expression vec- tor and the vector was induced to express recombinant fusion protein pd20 - TNF -α Objective To purify the fusion protein pd20 - TNF-α, then the purified protein is identified, and the biological activity of the protein pd20 - TNF -α is detec- ted. Methods Ni - NTA affinity chromatography was used to purify the fusion protein, than the fusion protein was detected by Western blotting. The effect of fusion protein pd20 - TNF -α against I929 cells was evaluated by MTT assay. Flow cytometry was used to detect early apoptosis rate of XGC9811 -L （a kind of high level hepatic metastasis from gastric cancer- related potential cell） which was affected by fusion protein with various concentration, this series experiments included 5 groups： A group （ pd20 - TNF -α 100.00 U/ml）, B group （ pd20 - TNF -α 25.00 U/ml）, C group （ pd20 - TNF -α 6. 25 U/ml）, D group （fusion protein was not added）, E group （apoptosis of KB by flow cytometry） . Cell invasion experiment was used to detect im- pact of fission protein on migration ability of XGC9811 -L, this series experiments included 4 groups： pd20 -TNF -α group, TNF -α group, XGC9811 - L group and control peptide group. Results By Ni - NTA affinity chromatography, the purified target fusion protein pd20 -TNF -αwas obtained when concentration of imidazole reached 200 mmol/L, the purity of protein was 92%. The purified protein had the ability of uniting with anti - TNF -αMeAb. The effect of pd20 - TNF-α against L929 cells which was evaluated by MTF assay indicated that the purified protein pd20 - TNF -α had strong ability of killing L929 cells, the killing activity was 7.6 x 106 U/mE The flow cytometry showed that there were significant differences in early apoptosis rates a- mong5 groups of cells [A, B, C, D, Egroup： （9.04±0.08）%, （6.96±1.21）%, （5.99~0.02）%, （0.73±0.02）%, （0. 01±0. 00）% ; F =2. 57, P 〈0. 05] . Cell invasion experiment results indicated that there were significant differences in pen- etrating cell number per unit time among pd20 -TNF-α group, TNF -ct group, XGC9811 -L group and control peptide group [ （26.5±5.9）, （36.0±3.2）, （63.5±5.0）, （64.0±6.8）; F=49.87, P〈0.01~ .Conclusion The fusion protein pd20 - TNF -α was purified successfully in this study, and the obvious biological activity of pd20 - TNF -α was proved. It has a- bility of increasing the early apoptosis rate of gastric cancer and inhibiting the invasive ability of gastric cancer cells.