DNA-PKcs单链抗体DPK3-scFv的原核表达纯化及生物学作用研究

Prokaryotic expression,purification and biological effect of the scFv against DNA-PKcs

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 study prokaryotic expression,purification of the single-chain antibody DPK3-scFv against DNA-PKcs,and to observe its interference with the phosphorylation of DNA-PKcs in response to ionizing radiation.Methods DPK3-scFv gene produced by PCR was inserted into the prokaryotic expression vector pET28a with His tag.The recombinant plasmids were transformed into bacteria strain BL21,and the expression conditions were optimized.The transmembrane and biological activity of DPK3-scFv were assessed by immunofluorescence microscope observation and Western immunoblotting.Results The recombinant prokaryotic expression vector of pET28a-DPK3-scFv was constructed.Optimized expression of soluble DPK3-scFv was obtained in E.coli BL21 cultured in 2xYT medium with 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside（IPTG） induction at 28℃.The purified DPK3-scFv could effectively inhibit the kinase activity of DNA-PKcs in vitro.The results of immunofluorescence observation and Western immunoblotting indicated that this recombinant scFv could enter cells and colocalize mainly with DNA-PKcs in the nuclei.DPK3-scFv significantly inhibited DNA-PKcs expression and abolished the phosphorylation of DNA-PKcs/pS2056 in response to γ-ray irradiation.Conclusion The soluble expression of DPK3-scFv antibody is obtained in E.coli BL21.DPK3-scFv can enter nuclei and has biological effect on inhibiting the kinase activity of DNA-PKcs.