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目的原核表达和纯化DNA依赖性蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)单链抗体DPK3-scFv,观察其透入细胞及干扰辐射诱发DNA-PKcs磷酸化修饰的生物学作用。方法 PCR扩增DPK3-scFv基因,插入到带有His标签的原核表达载体pET28a,重组质粒转化工程菌BL21、优化表达。免疫荧光显微镜和蛋白免疫印迹观察DPK3-scFv透膜进入HeLa细胞及对电离辐射诱发靶分子DNA-PKcs的磷酸化修饰的影响。结果成功构建单链抗体表达载体pET28a-DPK3-scFv,在2xYT培养基(16 g胰蛋白胨、5 g酵母提取物和10 g NaCl溶于1 L双蒸水中,高压灭菌)、20℃温度、0.2 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(isopropy1-β-D-thiogalactopyranoside,IPTG)条件下诱导可溶性表达,进一步获得了纯化单链抗体。DPK3-scFv能体外抑制DNA-PKcs激酶活性,纯化的DPK3-scFv可跨膜进入细胞内,并与DNA-PKcs共定位在细胞核。DPK3-scFv对γ射线照射HeLa细胞中DNA-PKcs及其S2056位点(pS2056)的自磷酸化具有显著抑制作用。结论通过优化条件获得了可溶性原核表达的DPK3-scFv单链抗体,具有抑制其靶分子DNA-PKcs的磷酸激酶活性。
Objective To prokaryotic express and purify the DNA-dependent protein kinase catalytic subunit (DPK3-scFv), which is a single-chain antibody against DNA-PKC, Learning role. Methods The DPK3-scFv gene was amplified by PCR and inserted into the prokaryotic expression vector pET28a with His tag. The recombinant plasmid was transformed into BL21 and optimized for expression. Immunofluorescence microscopy and Western blotting were used to investigate the effect of DPK3-scFv on HeLa cells and the phosphorylation of DNA-PKcs induced by ionizing radiation. Results The single-chain antibody expression vector pET28a-DPK3-scFv was constructed successfully and was autoclaved in 2xYT medium (16 g tryptone, 5 g yeast extract and 10 g NaCl in 1 L double distilled water), 20 ℃ temperature, Soluble expression was induced by 0.2 mmol / L isopropy1-β-D-thiogalactopyranoside (IPTG), and the purified single chain antibody was obtained. DPK3-scFv can inhibit DNA-PKcs kinase activity in vitro. The purified DPK3-scFv transmembrane into the cell and co-localized with DNA-PKcs in the nucleus. DPK3-scFv significantly inhibited the autophosphorylation of DNA-PKcs and its S2056 site (pS2056) in γ-irradiation HeLa cells. Conclusions Soluble prokaryotic DPK3-scFv single-chain antibody was obtained by optimizing the conditions and inhibited the phosphorylase activity of its target molecule DNA-PKcs.