抗肿瘤多肽TAT-N15p55PIK表达和纯化方法的改进及其应用

Improvement of Methods on Expression and Purification of Anti-tumor Peptide TAT-N15p55PIK and its Application

[目的]改进原核蛋白纯化方案,纯化抗肿瘤多肽TAT-N15p55PIK。[方法]通过SDS-PAGE、考马斯亮蓝染色和灰度扫描,筛选纯化蛋白最适合的菌株、确定目的蛋白浓度和纯度的准确检测方法,以及最佳诱导浓度。以咪唑洗脱,PBS透析和去除内毒素等方法,研究融合蛋白最佳纯化方案。以细胞免疫荧光和5-溴脱氧尿嘧啶核苷掺入法检测经纯化的融合蛋白对间质肿瘤细胞增殖的影响。[结果]筛选出最适蛋白表达菌株;确定1mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)为最佳的蛋白诱导表达浓度;以BCA法和SDS-PAGE灰度扫描相结合的方法测定纯化蛋白的浓度及纯度;通过改进后的纯化步骤,融合蛋白纯度从36%提高为61%,浓度从0.72μg/μl提高为1.5μg/μl,可100%转导入Hela细胞,转导入T淋巴细胞白血病细胞Jurkat48h能够显著抑制细胞增殖,抑制率为20.31%(P<0.01)。[结论]经纯化的TAT-N15p55PIK具有一定的抑制间质肿瘤细胞增殖的能力,为融合蛋白TAT-N15p55PIK的抗肿瘤临床应用奠定了重要的工作基础。

[Purpose] To improve the method of prokaryotic protein purification,to purificate anti-tumor peptide TAT-N15p55PIK. [Methods] The best strain for protein purification,the accurate method of testing concentration and purity of fusion prutein and the best concentration of inducing fusion protein ex-ression were ascertained by SDS-PAGE,Coomassie brilliant blue staining and gray scale scanning. A better method tbr protein purification was established by adding procedures of elution with Imidazole,PBS dialysis and endotoxin clean. Effeetions of TAT-fusion protein on proliferation of mesenchymal neoplasm were measured with cell immunofluorescence staining and Brdu incorporation assay. [Results] The beststrain for protein purification was selected. The fusion protein was the best induced with IPTG 1 mmol/L. The combination of BCA protein assay,SDS-PAGE and gray level scanning was established as a measure- ment to test protein concentration and purity. Through the new method of purification,the purity of fusion protein raised from 36% to 61% and the concentration of it increased from 0.72μg/μl to 1.5μg/μl. It can transduced into Hela cell line with 100% ratio detected by cell immunofluorescence staining. When transduced into T lymphocyte leukemia cell line Jurkat for 48h,it can markedly inhibited cell prolifera- tion with a ratio of 20.31%. [Conclusion] The purified fusion peptide TAT-N15p55PIK has some func- tions in inhibiting mesenchyme neoplasm cell proliferation. It constituted an important basis for its clini- eal application on anti-tumor therapy.